For screening the high-resolution system of microorganism and the application of other high-flux microorganismsSystem, kit, device and methodCross reference to related applications
The application be U.S. Non-provisional Patent application the 15/135,377th part being submitted on April 21st, 2016 afterContinuous application, it is required that in 2 months 2016 U.S. Provisional Patent Applications submitted for 24th the 62/299th, 088, in 2 months 2016 5Day submit U.S. Provisional Patent Application the 62/292,091st and in the U.S. Provisional Patent Application submitted on April 21st, 2015No. 62/150,677 benefit of priority.The application is also required in the U.S. Provisional Patent Application submitted on October 19th, 2016No. 62/410,377 benefit of priority.Each of these temporary patent applications are incorporated herein by reference in their entirety.
Sequence table is incorporated to
The application includes the sequence table submitted in ascii by EFS-Web, entitled " GALT_009_PCT_ST25.txt ", the size of this document are 3KB and generate on October 17th, 2017.The content of sequence table is whole by referenceIt is incorporated herein.
Technical field
The disclosure relates generally to the innovations of microbiology, micro Process, chemistry, optics, robot and information technology.MoreBody, this disclosure relates to be for high-throughput culture, screening, separation, sampling and/or identification biological entities and/or nutrientsSystem, equipment, kit and method.
Background technique
Traditional technology and tool for cultivating the biological entities from environment He other samples is usually slow, laborious and highIt is expensive.Even if cell and other biological entity still cannot usually cultivate success using these technologies and tool, cause information and/Or the missing of product chance.Equally, screening is used for specific metabolite, enzyme, protein, nucleic acid, phenotype, mutation, metabolic pathway, baseCause, adaptability, the biological entities group of ability and/or curative effect are challenging, need complicated and expensive method.For example, micro- lifeObject is lived in the extremely environment of high risk.In order to survive, microorganism has been developed that a set of wonderful biochemical tool, packetInclude new enzyme, unique metabolite, innovation genetic approach, and manipulate the strategy of its environment and microorganism neighbours, theseStrong solution can bring new opinion and product (from the antibiotic of rescue life to improvement food production and safetyFertilizer).
Invention summary
According to some embodiments, the disclosure is provided for simplifying culture process, supporting high flux screening and/or exploitation newOpinion and product microbiology system, equipment, kit and the method according to some embodiments.For example, equipment can be withIncluding for receiving the micromachining device for containing the sample of one or more cells.Micromachining device is for cultivating one or moreThe high density microwell array (being known as " hole " sometimes for simplifying) of a cell.
In some embodiments, the device with high density microwell array for cultivating cell can be provided.IntoIn the embodiment of one step, one group can be provided for being put into the unique label of each micropore or some micropores.Alternatively, described deviceThe unique label being inoculated with into each micropore or some micropores can be provided with.Device and/or unique label can be provided separately,Or a part as kit provides.For example, kit may include for receiving the sample for containing one or more cellsMicromachining device, the micromachining device is the high density microwell array for cultivating one or more cells.Kit is alsoIt may include one group for being put into the unique label of micropore, to identify the specific micropore for wherein cultivating cell category.It can be in heightOne or more unique labels in each micropore of density microwell array.Kit, which may further include, has uniqueness for being inoculated withThe micropore of label and/or tracked using unique label culture cell return to culture they specific micropore specification.
In some embodiments, unique label may include micro- with target specificity nucleotide sequence and for identifyingThe nucleic acid molecules of the location specific nucleotide sequence in hole itself, the target specificity nucleotide sequence be used for there may beThe target nucleic acid fragment of the particular types (such as organism) of cell or cell in micropore is annealed.It can be put in specific microporeEnter the unique label more than one, so that there is the location specific nucleotide sequence more than one in the specific micropore.That is,Location specific nucleotide sequence can be found (for example, predefining to identify high density micropore in the micropore more than oneThe dimension of micropore in array), and unique label can be multidimensional.For example, one group of unique label can be it is two-dimensional, including toolThere are the label of first position specific nucleotide sequences and the label with second position specific nucleotide sequences, predefinesThe first position specific nucleotide sequences are to identify the micropore (for example, row) of the first dimension in high density microwell array, in advanceFirst determine the second position specific nucleotide sequences with identify the second dimension in high density microwell array micropore (for example,Column).First position specific nucleotide sequences and second position specific nucleotide sequences identify high density microwell array togetherSpecific micropore.Therefore, in specific micropore the combination operation of unique label to identify the micropore.
In some embodiments, it can provide at device (including the high density microwell array for cultivating cell)Each of or some micropores in grow and/or screening cell one or more nutrients.One or more nutrition can be providedObject is used to be put into all or some micropore, such as is directly placed into micropore, by film and/or in permeability or semi-permeable film.Alternatively, described device, which can be provided with, has been placed in one of each micropore or some micropores or a variety of nutrients.Nutrients canA part offer to be provided separately, as culture medium, and/or a part offer as kit.Kit may includeFor providing the specification of one or more nutrients for the cell cultivated in the specific micropore of device.For example, kit canTo further comprise at least one nutrients for cultivating at least one cell of at least one type.For cultivating at least oneAt least one nutrients of at least one cell of a type may include or: at least one from least one type is thinThe ingredient of the extract of the natural surroundings of born of the same parents, the culture medium of the natural surroundings of at least one cell from least one type,The culture medium similar with the natural surroundings of at least one cell of at least one type prepared, only at least one is planted described in cultureThe Selective agar medium of at least one cell of class, and/or distinguish the identification training of at least one cell of at least one typeSupport base.The natural surroundings of at least one cell of at least one type can be biological tissue, biological product, microbial suspensionLiquid, air, soil, deposit, living organism, feed, petroleum, sewage and/or water.
In some embodiments, it can provide for cell to be maintained at device (including for cultivating the highly dense of cellSpend microwell array) each of or some micropores in film (for example, preventing cross contamination).Some or all can be sealed with filmMicropore.Alternatively, described device can be provided with the film of some or all sealed micropores.Film for example can be impermeable, only gas-permeable, or one or more nutrients is allowed to diffuse to one or more micropores.Device and/or film can be withIt is provided separately, or a part as kit provides.For example, kit may include containing at least one cell for receivingSample micromachining device.After kit can also be included in sample addition micromachining device, at least one cell is retainedFilm at least one micropore of high density microwell array.Kit may further include in micropore be added sample and/Or the specification that will be cultivated cell using film and be maintained in their specific micropore of culture.
In some embodiments, it provides using having the device of the high density microwell array for cultivating cell and cultivatesThe step of cell.For example, method may include that obtain the high density that defines for cultivating at least one cell from sample micro-The micromachining device of hole array, may include at least one unique label is put into high density microwell array at least one is micro-The sample including one or more cells is added in hole in a device, and/or to all or part of device application film to protect cellIt stays in its respective micropore.In some embodiments, sample is prepared before addition.For example, can with dilute sample so thatNo more than one cell is put into each micropore of most of high density microwell arrays.Method can also include incubating device withMultiple cells are grown from least one cell at least one micropore of high density microwell array.
In some embodiments, it discloses for replicating and/or being segmented in the cell cultivated in high density microwell arrayThe step of, allow to modify and/or destroy a part of cell for analyzing (for example, amplification and sequencing), and can retain surplusPurposes of the remaining cell for the sampling in future and/or based on the information obtained from analysis.For example, method may include by high densityMultiple cell segmentations at least one micropore of microwell array at first part multiple cells and second part it is multiple thinBorn of the same parents.At least one unique label can be kept together with multiple cells of at least first part.Divide high density microwell arrayMultiple cells at least one micropore include removing film from least one micropore of high density microwell array, so that some cellsIt is retained at least one micropore of high density microwell array, and some cells are retained on film.Alternatively, film can temporarily be shelledIt is sampled to allow to sample or pass through (that is, perforation).
In some embodiments, it discloses for analyzing all or some cell cultivated in high density microwell arrayThe step of.For example, method may include analyzing multiple cells of first part to determine at least one targeted species.To be analyzedCell may be at cultivating in their same micropore, be attached to the film of their micropore of covering culture, and/or be transferred to anotherA position (for example, culture dish or second device with corresponding high density microwell array).Analysis may include measuring at leastThe existence or non-existence of one targeted species, this can be based at least one nucleic acid and gene selects.For example, can be polymerizeEnzyme chain reaction (PCR), the target specificity nucleotide sequence based at least one unique label is come present in amplifying cellsAny target nucleic acids segment.Analysis may further include using next-generation sequencing (NGS) or any other sequencing type to appointingThe step of target nucleic acids segment of what PCR amplification is sequenced.Sequencing can be used and come identification of cell type (for example, organism)The one or more positions in the initial high density microwell array of culture cell category and/or.
In some embodiments, it discloses and wherein cultivates the highly dense of particular kind of cell for identifying and/or positioningThe step of spending one or more micropores of microwell array.For example, method may include identifying it based at least one unique labelAt least one micropore of the middle high density microwell array for cultivating at least one targeted species.Based on to wherein cultivating at least one meshThe identification of at least one micropore of the high density microwell array of type is marked, method may include in multiple cells of second partPosition at least one targeted species.
In some embodiments, it discloses for choosing or sampling in the high density microwell array for cultivating multiple cellsThe step of one or more micropores.For example, method may include choosing from specific micropore or film location relevant to specific microporeAt least one cell.Describe for from more than a micropore and meanwhile choose device and method.
In some embodiments, it discloses using having the device of the high density microwell array for cultivating cell and sievesThe method for selecting cell.For example, method may include screening the cell cultivated in high density microwell array to measure targeted speciesExistence or non-existence.If it does, method may further include chooses one from one or more micropores of culture cellOr the cell of multiple targeted species.Method may further include metabolin, metabolic pathway, enzyme, protein, core based on cellAcid, phenotype, gene, mutation, adaptability and/or ability are tested.If method may further include using unique labelOne or more micropores of wherein training objective type are identified using one or more location specific nucleotide sequences.ExampleSuch as, target nucleic acids segment is likely to be present in targeted species, and target nucleic acids segment includes being related to metabolin, metabolic pathway, enzyme, eggWhite matter, nucleic acid, phenotype, gene, mutation, adaptability and/or ability genetic code.
In some embodiments, it discloses using having the device of the high density microwell array for cultivating cell and surveysThe method of the relative abundance of targeted species and/or absolute abundance in random sample product.For example, method may include preparing sample and by sampleProduct are added thereto in the micropore equipped with one group of unique label, prepare each micropore that sample makes most of high density microwell arraysIn equipped at least one cell be no more than a cell.After culture, method may include measuring to have at least using labelFirst quantity of the micropore of one cell, the second quantity of the micropore with targeted species, and measured in sample at least based on thisThe relative abundance of one targeted species.Method may further include total number of cells of the measurement from sample, and be surveyed based on sumIn random sample product in the relative abundance of at least one targeted species and sample at least one targeted species absolute abundance.
In general, it discloses using the micromachining device for the high density arrays for defining experimental considerations unit and cultivates from sampleBiological entities system, equipment, kit and method.Biological entities can be organism (such as microorganism, such as bacterium orFungi), cell (such as eukaryocyte), cell component, cellular products and virus.It in some embodiments, is more than a kind of lifeObject entity can reside in sample, adding apparatus and/or addition experimental considerations unit.Experimental considerations unit may include one or more lifesObject entity and/or one or more unique labels are to identify spatial information relevant to particular experiment unit.Unique label can be withIncluding binding molecule, such as protein, nucleic acid, carbohydrate, lipid and/or drug.For example, unique label may include usingThe target specificity ingredient of the biomolecule present in combining target biological entities.Unique label can also include for identifyingThe location specific ingredient of spatial information relevant at least one experimental considerations unit.It can be with incubating device in the height of experimental considerations unitMultiple biological entities are grown in density array.Based at least one unique label, the biology of one or more cultures can analyzeEntity is to identify that one or more types and/or space relevant to wherein culture one or more experimental considerations units of entity are believedBreath.
In some embodiments, it is (such as bacterial cell, true that a kind of at least one of screening sample biological entities are providedNucleus) method.The method uses micromachining device as described herein, has the top surface for defining microwell array.At least one micropore of microwell array is added: (a) at least one cell from sample；(b) indicator substance；(c) a certain amount ofNutrients.Cell, indicator substance and nutrients can sequentially add in any order, or can be by any combination of themSequentially or simultaneously it is added.Film is applied to micromachining device so that at least one cell to be retained at least one micropore.It will be micro-Processing unit (plant) is incubated for a period of time in predefined conditions, with multiple thin from least one cell growth at least one microporeBorn of the same parents.The optical characteristics of at least one micropore, such as color, fluorescence, phosphorescence or luminous can be evaluated.The assessment can be incubated forAt when and/or multiple time points carry out, such as after micropore content is added but before incubation and incubation openedAfter beginning but not yet complete etc..Compare based on the optical characteristics assessed or repeatedly between optical characteristics evaluation, can determinePresence or absence of at least one target biological entities at least one micropore.
In some embodiments, the optical characteristics of at least one micropore is related to the amount of indicator substance or form.
In some embodiments, indicator substance includes inorganic phosphate not soluble in water.In these embodimentsIn some, target biological entities may include the microorganism for dissolving inorganic phosphate.
In some embodiments, indicator can be pH sensitive dye.For example, pH sensitive dye can be in selected pHChange color in visible-range in range.Alternatively, pH sensitive dye can within the scope of the first pH luminescence generated by light (fluorescence orPhosphorescence) and within the scope of the 2nd different pH will not luminescence generated by light, or luminescence generated by light is changed at different pHDyestuff.
In some embodiments, at least one target biological entities may include the microorganism for producing acid.
In some embodiments, measurement optical characteristics, which is included in during incubation, carries out multiple optics spy in different timeThe measurement of property.
In some embodiments, the method also includes determining that target biological entities are present at least one microporeIn the case where, at least some of multiple cells are transferred to target position after incubation.
In some embodiments, at least one micropore includes multiple micropores, and at least one cell packet is wherein addedInclude average one cell of addition into each of multiple micropores.
In some embodiments, each micropore of microwell array has about 25 μm to about 500 μm of diameter.In some realitiesIt applies in scheme, the superficial density of microwell array is at least 750 micropore/cm2。
In some embodiments, evaluation optical characteristics include: (a) be added at least one cell, indicator substance andThe optical characteristics of at least one micropore is measured after nutrients and before incubation；(b) it is micro- that at least one is measured after incubationThe optical characteristics in hole；With (c) compare before being incubated for and the optical characteristics that measures after being incubated for.
In some embodiments, it provides a kind of using having the micromachining device for the top surface for defining microwell array to sieveThe method of at least one of sampling product target biological entities.The described method includes: adding at least one micropore of microwell arrayEnter: (a) at least one cell from sample；(b) indicator substance；(c) a certain amount of nutrients；Film is applied to micro ProcessDevice is at least one cell to be retained at least one micropore；When micromachining device being incubated for one section in predefined conditionsBetween, with from the multiple cells of at least one cell culture at least one micropore；With the change based on indicator substance after incubationChange, determines at least one micropore presence or absence of at least one target biological entities.
In some embodiments, it provides a kind of using having the micromachining device for the top surface for defining microwell array to sieveThe method of at least one of sampling product target biological entities.The described method includes: indicator substance is saved in the more of microwell arrayIn each of a micropore；Sample is added to micromachining device, so that at least one micropore in multiple micropores connectsReceive at least one cell and a certain amount of nutrients from sample；Film is applied to micromachining device, institute is received extremelyA few cell and nutrients are retained in each of multiple micropores；Micromachining device is incubated for one section in predefined conditionsTime；With based on the optical characteristics for measuring at least one micropore, determine at least one micropore of the multiple microporePresence or absence of at least one objective microbe biological entities.
It should be understood that, it is contemplated that above-mentioned concept and other concepts discussed more fully below are (on condition that these concepts mutual oneCause) all combinations be invention disclosed herein theme a part.Specifically, it is contemplated that protected in the requirement that disclosure end occursAll combinations of the theme of shield are a part of invention disclosed herein theme.It should be appreciated that the term clearly used hereinIt can appear in any disclosure being incorporated by reference into, should be endowed most consistent with specific concept disclosed hereinMeaning.
By checking that the following drawings and detailed description, other systems, method and feature to those skilled in the art willIt is obvious.All these others systems, method and feature are intended to be included in this specification, in the scope of the present inventionIt is interior, and protected by the attached claims.
Detailed description of the invention
It will be understood by those skilled in the art that attached drawing being mainly described property purpose, and it is not intended to limit invention described hereinThe range of theme.The drawings are not necessarily drawn to scale；Under some cases, the various aspects of invention disclosed herein theme may beIt is exaggerated in attached drawing or amplifies display to promote the understanding to different characteristic.In the accompanying drawings, similar reference character generally refers to classAs feature (for example, function similar and/or the similar element of structure).
Fig. 1 is the perspective view for illustrating micromachining device or chip according to some embodiments.
Fig. 2A -2C is the top view of dimension for illustrating the micromachining device or chip according to some embodiments, side respectivelyView and end-view.
Fig. 3 A and 3B are illustrated according to the micromachining device of some embodiments or the decomposition view of chip and top view respectivelyFigure.
Fig. 4 A and 4B are the schematic diagrames for illustrating the film according to some embodiments.Fig. 4 C is that had according to some embodimentsThe image of the film surface for the trace to be formed is contacted with hole array.
Fig. 5 A is the flow chart illustrated according to some embodiments from the cellifugal method of sample point.Fig. 5 B is to illustrate basisSchematic diagram of some embodiments from the cellifugal method of pedotheque point.
Fig. 6 is the flow chart for illustrating to separate and cultivate from sample according to some embodiments the method for cell.
Fig. 7 is the schematic diagram for illustrating to separate and cultivate from complex sample according to some embodiments the method for cell.Small figure716 show output: cultivating the isolated bacterial strain (SEQ ID NO.2-6) of cell.
Fig. 8 A-8C is the schematic diagram for illustrating to be chosen according to some embodiments by a needle or multiple needles.
Fig. 9 A-9D is to show the image that hole is chosen according to some embodiments.
Figure 10 A-10D is the schematic diagram for illustrating to be used to choose according to some embodiments the tool of chip.
Figure 11 is the image in the hole chosen by thin layer agar, illustrates to pass through film or sealing according to some embodimentsThe selection of layer.
Figure 12 is the schematic diagram for illustrating the cross section of the chip 1200 according to some embodiments.
Figure 13 is the flow chart illustrated according to some embodiments for the method for screening.
Figure 14 is the schematic diagram for illustrating the screening technique according to some embodiments.
Figure 15 is a series of image of explanations according to the screening example of some embodiments.
Figure 16 A-16C is the image illustrated according to some embodiments from screening recycling.
Figure 17 A is the exploded view for illustrating the chip for screening according to some embodiments.Figure 17 B is according to some realitiesThe fluorescent image of chip after applying option screening.Figure 17 C is to show according to some embodiments, chooses sample from chip after screeningThe image of the process of product.
Figure 18 is the flow chart for illustrating the method for counting according to some embodiments.
Figure 19 is the schematic diagram for illustrating the method for counting according to some embodiments.Small Figure 191 6 shows output: sequenceWith the relative abundance (SEQ ID NO.2-6) of culture cell.
Figure 20 is the schematic diagram for illustrating the directory system according to some embodiments.
Figure 21 A-21E is the schematic diagram for illustrating the chip for having hole specific chemical object according to some embodiments.
Figure 22 A and 22B are the images for showing the chip of the high flux screening result according to some embodiments.
Figure 23 is the micrograph of the sample of the binary mixture in the addition to chip shown according to some embodimentsPicture.
Figure 24 A shows composograph, by one of the inverted microscope acquisition from chip (appropriate to have storage cavern and film)Image series are stitched together.Figure 24 B is the close-up image obtained from inverted microscope, which show the micropore of some growths andThe micropore of some skies.
Figure 25 shows the microscope figure for each micropore observed for pure culture (SC, AC and their mixture)Picture.
Figure 26 shows the image of the pure culture (in Figure 26 A) of SC and the pure culture (in Figure 26 B) of AC.
Figure 27 shows the gel electrophoresis result in the library NGS by embodiment test compilation according to some embodiments.Swimming lane1, the amplicon from simulation group gDNA；Swimming lane 2, the amplicon from simulation group gDNA；Swimming lane 3, negative control；Swimming lane4, DNA ladder shape band.
Figure 28 shows the classification of the NGS data collected by embodiment test according to some embodiments.
Figure 29 shows the gel electrophoresis result of PCR amplification from embodiment test according to some embodiments.SwimmingRoad 1: ladder-tape.Swimming lane 2-4: simulation group's bacterial 16 S rRNA amplicon.Swimming lane 5PCR negative control.
Figure 30 illustrates the process that bacterium disengaged position mark is carried out according to the double indexes of use of some embodiments.
Figure 31 is shown to be analyzed according to the NGS data in each hole from single 2,500 microwell chips of some embodimentsAs a result.
Figure 32 A-32D shows the micro-image of the micropore of the screening test of the phosphate solubilizing microorganism according to some embodiments.
Detailed description of the invention
The disclosure relates generally to for separating, cultivating, adaptability, sampling and/or screening biological entities and/or nutrientsSystem, kit, device and method.It discloses for receiving comprising at least one biological entities (for example, at least one is thinBorn of the same parents) sample micromachining device (or " chip ").Term " biological entities " can include but is not limited to organism, cell, thinBorn of the same parents' ingredient, cellular products and virus, term " type " can be used for describing a taxon, including but not limited to activity classificationUnit (OTU), genotype, phylotype, phenotype, the ecotype, history, behavior or interaction, product, variant and evolution are upper significantUnit.
Cell can be Archimycetes, bacterium and eucaryote (such as fungi).For example, cell can be microorganism, such asAerobic, anaerobism or facultative aerobic microbiological.Virus can be bacteriophage.Other cell component/products can include but is not limited toIt is protein, amino acid, enzyme, polysaccharide, atriphos (ATP), lipid, nucleic acid (such as DNA or RNA), nucleosides, nucleotide, thinAfter birth/cell wall, flagellum, pili, organelle, metabolin, vitamin, hormone, neurotransmitter and antibody.
Nutrients can be determining (such as culture medium chemically determine or synthesis) or uncertain (such as basisOr complex medium).Nutrients may include culture medium that either laboratory is prepared and/or that business manufactures (for example, two kindsOr the mixture of a variety of chemicals) ingredient.Nutrients may include either liquid nutrient media (i.e. nutrient broth), exampleThe ingredient of the raw bacterial context soup in such as sea, bacteriolyze meat soup (such as Luria meat soup).Nutrients may include either fluid nutrient mediumIngredient is mixed to form solid medium and/or commercially available manufacture agar plate, such as blood agar with agar.
Nutrients may include or the ingredient of selective medium.For example, Selective agar medium can be used for only growing it is certainBiological entities or the biological entities only with certain properties (such as antibiotic-resistant or the certain metabolins of synthesis).Nutrients can wrapIt includes or the ingredient of differential medium, is deposited by using specific indicator (such as dimethyl diaminophenazine chloride, phenol red, eosin Y or methylenum careuleum)Biochemical character under distinguishes a kind of biological entities and another biological entities or other kind of biological entities.
Nutrients may include or the ingredient of the extract from natural surroundings or culture medium.For example, nutrients can be withFrom the environment natural to certain types of biological entities, varying environment or multiple environment.Environment can include but is not limited to oneKind or a variety of biological tissues (such as connective tissue, muscle, nerve, epithelium, plant epidermis, blood vessel, elementary organization etc.), biology streamBody or other biological product (such as amniotic fluid, bile, blood, cerebrospinal fluid, earwax, exudate, fecal matter, gastric juice, interstitial fluid,Intracellular fluid, lymph, milk, mucus, rumen content, saliva, sebum, sperm, sweat, urine, vaginal fluid, vomitusDeng), microbial suspension, air (including such as gas with various content), supercritical carbon dioxide, soil (including such as mineralMatter, organic matter, gas, liquid, organism etc.), deposit (such as agricultural, ocean etc.), living organism (such as plantObject, insect, other small organisms and microorganism), dead organic matter, feed (such as herbage, beans, ensilage, crop residuesDeng), mineral, oil or oil product (such as animal, plant, petrochemical industry), water (such as natural fresh water, drinking water, seawater etc.)And/or sewage (such as health, business, industry and/or agricultural effluent and rainwash).
Micromachining device can define the high density microwell array for cultivating at least one biological entities.Term is " highly denseDegree " can refer to the ability that system or method are distributed many experiments in constant region domains.For example, including " high density " experimental considerations unitMicromachining device may include about 150 micropore/cm2To about 160,000 micropores or more/cm2, discussed further below.Other embodiments are shown in table 1.
Table 1
Micromachining device may include a series of substrate with functional layers.The functional layer of the series may include firstFunctional layer defines the experimental considerations unit (for example, hole) and at least one subsequent functional layer of the first array, defines aforementioned functionThe experimental considerations unit (such as micropore) of the subsequent array of each experimental considerations unit of layer.Each experimental considerations unit can be set to receive and trainSupport and/or screen biological entities and/or nutrients.Specifically, system as described herein, kit, device and method can be used forAutomation and/or high flux screening are directed to the different condition of high-density cells matrix.For example, system as described herein, kit,Device and method can be used for the effect tested and more one or more different nutrients grow microorganism, and/or screening generationThank object, enzymatic activity, mutation or other cell characteristics.
Fig. 1 is the perspective view for illustrating micromachining device or chip according to some embodiments.Chip 100 includes with micro-The substrate of mirror format slide forming, upper surface 102 have injection moulding feature.Feature includes four separated microwell arrays(microarray) 104 and injection label 106.Micropore in each microarray is arranged with grid pattern, in the edge of chip 100 weekEnclose has seamless edge between microarray 104.
Fig. 2A -2C is top view, side view and the end view for illustrating the dimension of the chip 100 according to some embodiments respectivelyFigure.In Fig. 2A, the top surface of chip 100 is about 25.5mm multiplied by 75.5mm.In Fig. 2 B, the end of chip 100 is about that 25.5mm multipliesWith 0.8mm.In Fig. 2 C, the side of chip 100 is about 75.5mm multiplied by 0.8mm.
It, can be at least part of micromachining device application film after micromachining device is added in sample.Fig. 3 A is from rootAccording to the decomposition view of micromachining device 300 shown by top view in Fig. 3 B of some embodiments.Device 300 includes accommodating exampleSuch as the chip of the array with hole 302 of edaphon.Film 304 is placed in above 302 array of hole.Gasket 306 is placed on film 304Side.Polycarbonate cap 308 with filling hole 310 is placed in 306 top of gasket.Finally, applying band 312 to lid 308.
Film can cover at least part of micromachining device, including one or more experimental considerations units, hole or micropore.ExampleSuch as, after micromachining device being added in sample, at least a film can be applied at least one micropore of high density microwell array.It canTo apply multiple films to the multiple portions of micromachining device.For example, can be applied to the individual branch of high density microwell arrayIndividual film.
Film can be connected, attachment, partially attachment, fixed, sealing and/or part are sealed to micromachining device, nearA kind of few biological entities are retained at least one micropore of high density microwell array.It is it is, for example, possible to use being laminated that film is reversibleGround is fixed to micromachining device.Film can be punctured, be removed, separated, is partially separated, removed and/or part is removed to obtain heightAt least one of at least one micropore of density microwell array biological entities.
A part of cell mass at least one experimental considerations unit, hole or micropore can be attached on film (via such as inhalingIt is attached).If so, the cell mass at least one experimental considerations unit, hole or micropore can be sampled by stripping film, so that at least oneThe part cell mass in a experimental considerations unit, hole or micropore is still attached on film.
Fig. 4 A and 4B are the schematic diagrames for illustrating the film according to some embodiments.Fig. 4 A shows the side view of chip 400,Define filled with content hole array and in hole array encapsulating chip 400 film 402 so that when from chip 400 removeWhen, the surface of the film 402 contacted with chip 400 with the trace in each hole and has in the hole of attachment (such as sticking) thereonTolerant sample, as shown in Figure 4 B.Fig. 4 C is that have the film table that the trace to be formed is contacted with hole array according to some embodimentsThe image in face.
Film can be it is the infiltration of impermeable, semipermeable, permselective, otherness and/or partial penetration,In at least one micropore to allow at least one nutrients diffusion high density microwell array.For example, film may include naturalMaterial and/or synthetic material.In some embodiments, the sufficiently small film in selection aperture will be will at least protect some or all cellsIt stays in micropore.For mammalian cell, aperture can be several microns, and still retain cell.However, in some embodiment partyIn case, aperture can be less than or equal to about 0.2 μm, such as 0.1 μm.Film diameter and aperture depend on material.It is, for example, possible to useHydrophily polycarbonate membrane, diameter can be about 10mm to about 3000mm, and aperture can be about 0.01 μm to about 30.0 μm.NoThe aperture of permeable membrane is close to 0.In the embodiment with impermeable membrane, any nutrients must provide before being sealed with filmIn micropore.Gas-permeable but the impermeable film of liquid may allow that oxygen enters micropore and carbon dioxide goes out from microporeIt goes.Film can have labyrinth, and aperture can determine or not know.However, hole can be nanoscale.Selective membrane itsHis factor may include cost, sealability and/or sterilization capability.
Substrate can define the micro channel array that the second surface opposite with the first surface is extended to from first surface.Microchannel can have the first opening in first surface and the second opening in second surface.It can be at least part ofThe first film is applied on one surface, so that at least some of at least one microchannel cell mass is attached to the first film.It can be at leastThe second surface of a part applies the second detachable film, so that at least some of at least one microchannel cell mass is attached to theTwo films.The cell mass at least one microchannel is sampled by removing the first film, so that at least one at least one microchannelA little cell masses are still adhered to the first film, and/or the second film of removing, so that at least some of at least one microchannel cell massIt is still adhered to the second film.
Term " high throughput " can refer to that system or method can be realized the quick execution of parallel or sequence many experimentsAbility.The example of " high throughput " system may include there is the automation equipment of cytobiology technology with prepare, be incubated for and/Or a large amount of chemistry, heredity, pharmacology, optics and/or image analysis are carried out, to screen one or more biological entities extremelyA kind of few metabolin, enzyme, protein, nucleic acid, phenotype, mutation, metabolic pathway, gene, adaptability and ability, as begged for hereinBy.According to some embodiments, " high throughput " can refer to that scale is at least about 96 experiments at least about 10,000,000While experiment or almost experiment simultaneously.
System disclosed herein, kit, device and method can be used for for biological entities (such as cell) matrix notWith the high flux screening of condition." Kong Zhongkong " concept can pass through manufacture (for example, micro Process) any need or desired levelSubstrate with multiple function layers or chip realize (that is, hole in hole in hole in hole).First functional layer can be withDefine experimental considerations unit (such as hole) array.Each experimental considerations unit is indicated by defining subsequent experimental considerations unit (such as micropore) arraySecond functional layer.This allows to carry out multiple experiments or test simultaneously on a single chip, to allow high-throughput operation.
For example, in figures 3 a and 3b, gasket 306 is placed in 304 top of film, the film 304 is applied to according to some embodiment partyThe array in the hole 302 on the micromachining device 300 of case.Gasket 306 only has an opening.However, in further embodiment partyIn case, it can be placed above device (with or without film) multiple compared with minipad or with more than one with smaller openingThe single gasket of a smaller opening, to form the functional layer being located therein or the larger experimental considerations unit battle array with subsequent functional layerColumn or subsequent experimental considerations unit array (such as hole 302).
Using multiple function layers, such as can simultaneously or almost simultaneously test more than a kind of nutrients or nutritional preparation.ExampleSuch as, same format can be used to screen the special ability of metabolin or cell or avoid making microbial compared with other nutrientsRely on nutrients derived from environment.
Experimental considerations unit is the predetermined site on micromachining device surface.For example, can be with the surface of design chips to consolidate cellIt is scheduled in the predetermined site of the first array.These predetermined sites can be hole, micropore, microchannel and/or specified fixation site.For example, surface can be manufactured to define microwell array.It can be by determining wall or addition wall by array part in substrate.ExampleSuch as, surface can be manufactured to define the hole of the first array first, wherein manufacturing the inner surface in each hole again to define second arrayMicropore, microchannel or fixed site.In another example, surface can be manufactured to define microwell array, answered to the surfaceWith another substrate (such as agar, plastics or another material) to be separated by the surface and by the micropore that it is defined.Each hole,Micropore, microchannel and/or fixed site can be set to receive and grow at least one cell；However, in use, it is any to giveFixed hole, micropore, microchannel or fixed site can actually receive or not receive and/or grow one or more cells.It is realThe type of verification certificate member is commutative.For example, the embodiment of micropore explicitly described herein is also intended to open wherein micropore at least partlyThe embodiment for replacing with microchannel, fixed site and/or other kinds of experimental considerations unit.
One or more parts of micromachining device can be selected with surface chemical modification agent, processing and/or coating are to haveThere is specific surface chemistry.For example, the substrate surface of at least one portion can be set to first surface feature or secondSurface characteristics, the first surface feature repel cell and/or reduce the tendency of cell adherence on the surface, the second surfaceFeature attracts cell and/or increases the tendency of cell adherence on the surface.According to the type of target cell, material and/or coating canTo be hydrophobic and/or hydrophilic.At least part top surface of substrate be can handle to repel target with first surface featureThe tendency of cell and/or the adherency of reduction target cell on the surface.Meanwhile it can handle each experimental considerations unit, hole or micropore at leastA part of inner surface occupies experimental considerations unit, hole or micro- to attract target cell and/or increase target cell with second surface featureThe tendency in hole.Substrate surface can have multiple portions, with different surfaces feature.
Chemical vapor deposition, electroporation, corona treatment and/or electrochemical deposition Applied Surface Chemistry can be used to changeProperty agent.Surface chemical modification agent can control surface potential, Lund current potential, zeta potential, configuration of surface, hydrophobicity and/or hydrophilicProperty.Surface chemical modification agent may include salt water, polyelectrolyte, metal, polymer, antibody and/or plasma.For example, tableFace chemical modifier may include octadecyl trichlorosilane alkane.Surface chemical modification agent may include dynamic copolymers, such as poly-Ethylene oxide (20) sorbitan mono-laurate and/or polyethylene glycol P (1,13,3- tetramethyl butyl)-phenyl ether.It comes to the surfaceLearning modifying agent may include static copolymer, such as poloxamer188, poly (L-lysine), and/or the poly- (L- of poly(ethylene glycol)-Lysine) block copolymer.
Equipment for screening the different condition for being directed to cellular matrices may include with the surface for defining microwell arraySubstrate.The part of microwell array is segmented into time array (such as by large hole or wall).Substrate can be with micro Process.Each microporeIt can receive and grow at least one biological entities (such as cell).The matrix of gained biological entities (such as cell) can be lifeThe high-density matrix of object entity.First array and/or second array can be plane, substantially planar and/or more planes(such as on idler wheel).
Term " high-resolution " can refer to that system or method distinguish the ability that can must largely test.For example, " high-resolutionThe system or method of rate " can select an experimental considerations unit from the micromachining device comprising high density experimental considerations unit, wherein describedThe diameter of experimental considerations unit is about 1nm to about 800 μm.The substrate of micromachining device or chip may include about or more than 10,000,000 micropore.For example, microwell array may include at least 96 positions, at least 1,000 position, at least 5,000 position,At least 10,000 positions, at least 50,000 positions, at least 100,000 positions, at least 500,000 positions, at least 1,000,000 position, at least 5,000,000 positions or at least 10,000,000 positions.
The superficial density of micropore is about 150 micropore/cm2To about 160,000 micropore/cm2Or it is higher.Micromachining deviceOr the micropore surface density of the substrate of chip is at least 150 micropore/cm2, at least 250 micropore/cm2, at least 400 micropores/cm2, at least 500 micropore/cm2, at least 750 micropore/cm2, at least 1,000 micropore/cm2, at least 2,500 micropores/cm2, at least 5,000 micropore/cm2, at least 7,500 micropore/cm2, at least 10,000 micropore/cm2, at least 50,000Micropore/cm2, at least 100,000 micropore/cm2Or at least 160,000 micropore/cm2。
The size of micropore can be from nanoscale (such as diameter is about 1 to about 100 nanometer) to micron order or bigger.For example,The diameter of each micropore can be 1 μm to about 800 μm, diameter be about 25 μm to about 500 μm or diameter be about 30 μm to about 100 μm.The diameter of micropore can be about or less than 1 μm, about or less than 5 μm, about or less than 10 μm, about or less than 25 μm, about or less than 50 μM, about or less than 100 μm, about or less than 200 μm, about or less than 300 μm, about or less than 400 μm, about or less than 500 μm, about orLess than 600 μm, about or less than 700 μm or about or less than 800 μm.
The depth of micropore can be about 500 μm to about 5000 μm, depth be about 1 μm to about 500 μm or depth be about 25 μmTo about 100 μm.The depth of micropore can be about 1 μm, about 5 μm, about 10 μm, about 25 μm, about 50 μm, about 100 μm, about 200 μm, about300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 1000 μm, about 1,500 μm, about 2,000 μm, about3,000 μm or about 5,000 μm.
The opening of each micropore or section can be arbitrary shape, such as round, hexagon or square.Each micropore can wrapInclude side wall.For not being circular micropore in its opening or section, the diameter of micropore as described herein refers to equal faceLong-pending circular effective diameter.For example, having equal areas, (100 is flat for the rectangular micropore of the side length with 10 × 10 micronsSquare micron) diameter of a circle be 11.3 microns.It can be put at least one micropore of high density microwell array following into oneAt least one unique location specific label of description is walked to promote identifying species and type and high density microwell arrayThe connection of specific micropore.At least one unique label can be put into and/or be placed in at least side of micropore bottom and/or micropore.At least one unique label may include nucleic acid molecules, has and moves back for the target nucleic acids segment at least one biological entitiesThe position of at least one micropore of the sum with target specificity nucleotide sequence of fire for identifying high density microwell array is specialSpecific nucleotide sequence.
For example, the substrate of micromachining device or chip can have having a size of from about 4 inches multiplied by 4 inches of surface.SurfaceThe array of about 100,000,000 micropores can be defined.Microwell array can be divided into about 100 subdivisions by wall and/or substrate can be with boundaryThe array in 100 holes is concludeed a contract or treaty, wherein a total of about 100,000,000 micropores of about 1,000,000 defined in each subdivision or hole micropore.It is testingUnder the service condition of different nutrients, can on chip microorganism of the loading from environmental sample so that single microbial orMicroorganism cluster is divided into the micropore of chip, and each micropore is located at the bottom of large hole.Each large hole may include experimental considerations unit, makeCan parallel on the same chip or about 100 kinds of different nutrients of sequential testing, wherein each hole provides up to 1,000,000 surveysTry example.
Target cell can be Archimycetes, bacterium and eucaryote (such as fungi, plant or animal).For example, target cell canTo be microorganism, such as aerobic, anaerobism and/or facultative aerobic microbiological.It can parallel on the composition of target cell or sequenceTest different nutrients are to analyze and more such as growth to cell mass, cell component and/or cellular products or other influences.It can screen cell component, product and/or the ability of target cell composition, such as one or more viral (such as bacteriophages),Cell surface (such as cell membrane or cell wall), metabolin, vitamin, hormone, neurotransmitter, antibody, amino acid, enzyme, albumenMatter, polysaccharide, ATP, lipid, nucleosides, nucleotide, nucleic acid (such as DNA or RNA), phenotype, mutation, metabolic pathway, gene and adaptationProperty.
Cell composition may include environmental sample extract and/or diluent.Environmental sample extract and/or diluentCan include but is not limited to one or more biological tissues (such as connective tissue, muscle, nerve, epithelium, plant epidermis, blood vessel,Elementary organization etc.), biofluid or other biological product (such as amniotic fluid, bile, blood, cerebrospinal fluid, earwax, exudate, excrementObject, gastric juice, interstitial fluid, intracellular fluid, lymph, milk, mucus, rumen content, saliva, sebum, sperm, sweat, urine,Vaginal fluid, vomitus etc.), microbial suspension, air (including such as gas with various content), supercritical carbon dioxide,Soil (including such as minerals, organic matter, gas, liquid, organism etc.), is lived at deposit (such as agricultural, ocean etc.)Organic matter (such as plant, insect, other small organisms and microorganism), dead organic matter, feed (such as herbage, beans, bluenessStore feed, crop residues etc.), mineral, oil or oil product (such as animal, plant, petrochemical industry), water (such as natural fresh water,Drinking water, seawater etc.) and/or sewage (such as health, business, industry and/or agricultural effluent and rainwash).
Before wrapping celliferous composition to micromachining device application (such as loading), method may include thin by combiningBorn of the same parents and environmental sample extract and/or diluent prepare composition.Method may further include liquefaction environmental sample extractAnd/or diluent.The concentration of cell in composition can be adjusted to the target of one each experimental considerations unit, hole or micropore cellDistribution.
If sample includes cell and/or virus, can be after applying to micromachining device by the cell cracking in sampleTo discharge nucleic acid molecules.It can be split with chemical treatment such as alkali exposure, detergent, ultrasonic wave, protease enzyme K or lysozyme exposureSolve cell.It can also be by heating lytic cell.
Fig. 5 A is the flow chart illustrated according to some embodiments from the cellifugal method of sample point.In step 500, it obtainsObtain sample.In step 502, at least one physical technique (such as mixing and/or ultrasonic treatment) and chemical technology (such as chelaMixture, detergent and/or enzyme) sample is homogenized and/or dispersed.In step 504, using for exampleIt is non-Granule medium (can obtain from Progen Biotechnik GmbH, Heidelberg, Germany) is separated equal by density centrifugationCell in matter and/or dispersion sample.
Fig. 5 B is the schematic diagram illustrated according to some embodiments from the cellifugal method of pedotheque point.Small Figure 50 6 showsPedotheque is gone out.Small Figure 50 8 shows the homogeneous in test tube and/or dispersion sample.After small Figure 51 0 shows centrifugationSample is divided into solvable fragment 512, cell 514,516 and of insoluble fragment518。
Fig. 6 is the flow chart for illustrating to separate and cultivate from sample according to some embodiments the method for cell.In step 600In, obtain sample.In step 602, from least one cell of gained sample extraction.In step 604, in micromachining device orThe cell of at least one extraction of loading at least one high density microwell array of chip.Step 604 may include at least oneThe cell preparation cell concentration object of a extraction selects at least one nutrients/culture medium, and/or a selection at least film.In stepIn rapid 606, at least part of microwell array is sealed so that cell concentration object to be retained in micropore with the film of at least one selection.In step 608, it is incubated for chip.Step 608 may include selection temperature, determine atmosphere (such as aerobic or anaerobism), and/or incubateEducate the time.In step 610, according to method described herein, chip is separated and/or is replicated and (uses such as Chooser), obtainedObtain the culture cell of two parts.For example, an at least film can be removed, so that a part culture cell still adheres to, or strippingFrom or puncture to sample culture cell.In operating procedure 612, a part culture cell is put to death for identifying.Step 612 can be withIt is analyzed including PCR, sequencing and/or various data.In step 614, aimed strain is identified.Can with such as aimed strain and/Or the culture cell of remainder is further cultivated, tested and/or is identified.
Fig. 7 is the schematic diagram for illustrating to separate and cultivate from complex sample according to some embodiments the method for cell.Small figure700 show the example of complex sample, especially micropopulation sample 702 and pedotheque 704.In small Figure 70 6, use exampleScheme as shown in Fig. 5 A and 5B is from least one cell of sample extraction.In small Figure 70 8, by the cell of at least one extraction(and any environmental extract and/or diluent) is loaded to micromachining device or core at least one high density microwell arrayPiece 710.Chip 710 and kit 712 can be put into couveuse 714.Reagent can be used for adding liquid with maintain growth and/orThe nutritional need of various screening purposes.Small Figure 71 6 shows output: cultivating the isolated bacterial strain of cell.
The type or hierarchical classification of the cell or microorganism that grow in identification micropore need skill including but not limited to belowArt: DNA sequencing, nucleic acid hybridization, mass spectrum, infrared spectroscopy, DNA cloning and antibody combine, to identify genetic elements or other typesIdentifier.Many identification methods and processing step kill microorganism, to prevent the further culture and research of objective microbe.In order to identification of cell or microorganism, while subsequent culture, research and the further exhaustive for allowing specific objective to clone,Design further embodiment is used to sample each experimental considerations unit, hole or the micropore on substrate or chip, while keeping testingThe locally completeness and separation of the micropopulation of unit, hole or micropore.
Substrate as described above can permit using further system, kit, device and method sampling cell mass.ExampleIt such as, can be with based first surface application selecting device.The apparatus may include at least one protrusions towards first surface.The diameter of at least one protrusion is less than the opening diameter of each micropore, hole or experimental considerations unit.At least one protrusion can be insertedEnter to accommodate at least one micropore, hole or the experimental considerations unit of cell mass, so that one at least one micropore, hole or experimental considerations unitDivide cell mass adherency and/or is attached at least one described protrusion.It can be taken out by removing device from the first surface of substrateCell mass sample at least one micropore, hole or experimental considerations unit, so that one at least one micropore, hole or experimental considerations unitDivide cell mass adherency and/or is attached at least one described protrusion.What each protrusion can be needle or multiple needles assembles object.
Fig. 8 A-8C is the schematic diagram for illustrating to be chosen according to some embodiments by a needle or multiple needles.Chip is provided800 choose for observing via microscope 802, and via control device 804 is chosen.In fig. 8 a, control device 804 is chosen to wrapContaining the arm 806 with single needle.In the fig. 8b, the arm 808 with multiple needles is shown.Fig. 8 C is the saturating of chip during choosingView.
Fig. 9 A-9D is to show the image that hole is chosen according to some embodiments.In figure 9 a, Kong Shiman.In figures 9 b and 9,Needle is moved in position.In Fig. 9 C, hole is chosen.In Fig. 9 D, sample is taken out from hole.
Figure 10 A-10D is to show the schematic diagram for being used to choose the tool of chip according to some embodiments.In Figure 10 A,Tool comprising multiple needles is aligned with the chip with multiple holes.In fig. 1 ob, reduction tool immerses needle in hole.?In Figure 10 C, the needle with attachment sample is shown, and sample is transferred to new chip.Alternatively, in figure 10d, quickly overturningTool is retained in sample with tool sheet.
Figure 11 is the image in the hole chosen by thin layer agar, illustrates to pass through film or sealing according to some embodimentsThe selection of layer.
Alternatively, when at least one protrusion is inserted at least one micropore, hole or experimental considerations unit, at least one described micropore,A part of population of cells in hole or experimental considerations unit accumulates upward displacement and surrounds at least one protrusion, so that at least some volumes are movedThe part of position is on the first surface of substrate and/or on the inner surface of at least one micropore, hole or experimental considerations unit.Method is also wrappedIt includes and cell mass is sampled at least one micropore by the cell mass for collecting at least some volume displacement parts.
Similar selecting device can be applied to the second surface opposite with the first surface of substrate.The apparatus may includeAt least one protrusion towards second surface.The diameter of at least one protrusion is approximately equal to or less than at least one micropore, holeOr the diameter of experimental considerations unit.At least one described protrusion is pressed against on the second surface and is accommodated described in cell mass at leastOne micropore, hole or the corresponding position of experimental considerations unit, and/or insertion accommodate at least one described micropore, the Kong Huo of cell massIn experimental considerations unit, so that a part of cell mass at least one micropore, hole or experimental considerations unit is moved to the first surface of substrateAnd/or on the inner surface of at least one micropore, hole or experimental considerations unit.Then the cell mass of movable part can be collected.Cell massIt can be located on the plug (such as hydrogel or other soft materials such as agar) at least one experimental considerations unit, hole or micropore, makeProper when at least one protrusion being pressed against second surface and being inserted at least one micropore, plug is moved to move describedPartial cell mass.
Cell mass sample from least one experimental considerations unit, hole or micropore can be placed in the second position.FurtherBefore sampling, at least one protrusion can be cleaned and/or be sterilized.In order to be conducive to cell attachment surface characteristics, at least oneAt least part of a protrusion can be made of the material for being handled and/or being coated with surface chemical modification agent.At least one protrusionIt can be array of protrusions.When based first surface application apparatus, the array of protrusions can be inserted corresponding experimental considerations unit,Hole or microwell array.Number of projection can correspond to the quantity of experimental considerations unit in the first array, one second in array of protrusionsIn microwell array in the quantity or substrate of micropore micropore sum.
Another device for sampling cell mass in substrate includes at least one needle and/or nanometer towards first surfaceSuction pipe.At least one needle and/or the overall diameter of nanopipette are less than the opening diameter of each micropore, and its interior diameter can accommodate targetCell dia.At least one needle and/or nanopipette insertion accommodate at least one experimental considerations unit, hole or the micropore of cell mass.A part of cell mass is taken out described at least one from least one described experimental considerations unit, hole or micropore introducing device using pressureCell mass sample in a experimental considerations unit, hole or micropore.
Cell mass sample from least one experimental considerations unit, hole or micropore can be placed in the second position.FurtherBefore sampling, at least one needle and/or nanopipette can be cleaned and/or be sterilized.At least one needle and/or nanopipette canTo be needle and/or nanopipette array.When to the first surface application apparatus of micro Process substrate, the needle and/or nanopipetteCorresponding experimental considerations unit, hole or microwell array can be inserted in array.Needle and/or nanopipette in needle and/or nanopipette arrayQuantity can correspond to the quantity of experimental considerations unit in the first array, in second microwell array in the quantity or substrate of microporeThe sum of micropore.
Another method for sampling cell mass in substrate includes at least one reality for accommodating the cell mass in liquidThe acoustic wave energy that verification certificate member, hole or micropore application focus.Can by effectively from least one micropore spray drop in a manner of applicationThe acoustic wave energy of focusing, such as sound drop injection (ADE) is (see, for example, Sackmann etc., " AcousticalMicro-and Nanofluidics:Synthesis,Assembly and Other Applications,"ProceedingsOf the 4th European Conference on Microfluidics (in December, 2014)).The drop may includeThe sample of cell mass at least one experimental considerations unit, hole or micropore.The drop can import second container or surface or substrate.
Substrate may include at least first and second, and described first includes at least part first surface, describedSecond includes at least part second surface.Described first and second along at least part and the first surface andThe parallel plane of second surface is detachably connected.The plane divides experimental considerations unit, hole or micropore.Pass through disassembly first and theTwo cell masses sampled at least one experimental considerations unit, hole or micropore, so that at least one experimental considerations unit, hole or microporeThe cell mass of first part is still adhered to first, and the second part at least one experimental considerations unit, hole or micropore is thinBorn of the same parents group is still adhered to second.
Figure 12 is the schematic diagram for illustrating the cross section of the chip 1200 according to some embodiments.Chip 1200 includes definingThe substrate of 1202 array of hole filled with content 1204.Substrate includes first 1206 and second 1208.First 1206With second 1208 along with 1202 array parallel of hole and divide the plane 1210 of 1202 array of hole equally and be detachably connected.When first1206 and when second 1208 disassembly, hole 1202 and its content 1204 are separated, and two parts of contents 1204 are caused, and retain coreThe isolation of content 1204 and position on piece 1200.
Each micropore, experimental considerations unit or microchannel may include partial barrier, by micropore, experimental considerations unit or channel portionIt is separated into first part and bottom part, cell is grown in both first part and bottom part.It is thin in samplingBefore born of the same parents group, the above method may include the cell cluster in dispersion and/or reduction cell mass.In dispersion and/or reduction cell massCell cluster can include but is not limited to using ultrasonic wave, oscillation and dispersed with little particle.
The above method, which may further include, is saved into from least one experimental considerations unit, hole or micropore for cell mass sampleTwo positions.The second position can be corresponding experimental considerations unit, hole or microwell array.The second position can be single container.It can be withRetain the cell mass sample from least one experimental considerations unit, hole or micropore for then cultivating.Alternatively, can retain from extremelyThe remaining cell of the cell mass of few an experimental considerations unit, hole or micropore is for then cultivating.
The above method may further include from the remaining cell of cell mass sample and/or cell mass and identify that at least one is thinBorn of the same parents.This may include carrying out DNA, cDNA and/or RNA amplification, DNA and/or RNA sequencing, nucleic acid hybridization, mass spectrum and/or antibodyIn conjunction with.Or or it is additional, this may include experimental considerations unit, hole or the micropore for identifying at least one cell origin.Packet can be usedThe unique label of the nucleotide sequence containing location specific marks each experimental considerations unit, hole or micropore.For identification experiment unit, Kong HuoMicropore can identify location specific nucleotide sequence, and location specific nucleotide sequence in sequencing and/or amplified reactionIt may be related at least one experimental considerations unit of at least one cell origin, hole or micropore.
Micromachining device as described above is able to use further system, kit, device and method culture from ringCell in the sample in border.For example, can be with based first surface application sample, so that at least one cell occupies at least oneA micropore, hole or experimental considerations unit.To at least part first surface (for example, at least part experimental considerations unit or the inner surface in hole)Using semi-permeable film, nutrients is allowed to diffuse at least one micropore, hole or experimental considerations unit.Meanwhile it preventing and/or subtractingGently cell is occupied to flee from from least one micropore, hole or experimental considerations unit.Semi-permeable film can be such as hydrogel layer.Use exampleSuch as lamination can be reversible by semi-permeable film or be irreversibly connected or fixed to substrate.Therefore, there can be at least oneIt is incubated at least one micropore of nutrients, hole or experimental considerations unit and occupies cell.It is exchanged using progressive part, cell can beOther at least one nutrients or nutritional preparation are gradually transitions from least one nutrients in a period of time, so that experience is tamed and dociledChange or adapts to.
The first nutrients incubation from environment can be used and occupy the thin of at least one first experimental considerations unit, hole or microporeBorn of the same parents, and the second nutrients from environment can be used and be incubated for the cell for occupying at least one the second experimental considerations unit, hole or micropore.The above method may include compare occupy at least one first experimental considerations unit, hole or micropore cell and occupy at least one secondThe cell of experimental considerations unit, hole or micropore, to analyze the first nutrients and the second nutrients.
For example, method may include one or more following steps:
Chip is obtained, 1000 to 10,000,000 or more micropores are defined in a large amount of large holes or fluidic cell, respectivelyThe diameter of micropore is about 1 μm to about 800 μm, and depth is about 1 μm to about 800 μm, and chip further has one or more settingsTo promote target microorganism to be moved to the surface chemistry of micropore；
To the chip application environment sample or the derivative of environmental sample, so that any target microorganism is located at microporeIn；
One or more semipermeability filter paper, hydrogel layer or other barriers are placed on chip, so that generating allowsNutrients diffuses into micropore but prevents and/or mitigate the barrier that microorganism is fled from from micropore；
Chip is incubated for at least one nutrients (for example originating from environment)；
By the exchange of progressive part gradually at least one other nutrients (such as preparation) replacement nutrition comeSource；With
Detect any growth of microorganism in micropore.
Target cell can be Archimycetes, bacterium or eucaryote.Target viral can be bacteriophage.When targeting virus,The micropore of chip can also include the host cell that can wherein grow virus.Detection occupies cell or the growth of virus and can wrapInclude detection following aspect variation: biomass (such as DNA/RNA/ protein/lipid), the existence or non-existence of metabolin, pH,Nutrients consumption and/or gas consumption.It may include carrying out real time sequence imaging, showing that detection, which occupies cell or the growth of virus,Micro mirror, optical density (OD), fluorescence microscope, mass spectrum, electrochemistry, amplification (DNA, cDNA and/or RNA), sequencing (DNA and/orRNA), nucleic acid hybridization and/or antibody combine.
Figure 13 is the flow chart illustrated according to some embodiments for the method for screening.In step 1300, sample is obtainedProduct.In step 1302, from least one cell of gained sample extraction.In step 1304, in micromachining device or chipThe cell of at least one extraction of loading at least one high density microwell array.Step 1304 may include being extracted at least oneCell preparation cell concentration object, select at least one nutrients/culture medium, and/or a selection at least film.In step 1306In, at least part of microwell array is sealed with the film of at least one selection cell concentration object to be retained in micropore.In stepIn rapid 1308, it is incubated for chip.Step 1308 may include selection temperature, determine atmosphere (such as aerobic or anaerobism), and/or be incubated forTime.It can carry out genetic screening and/or functional screening.In step 1310, to chip application genetic screening.In step 1312In, according to method described herein, chip is separated and/or replicated and (uses such as Chooser), obtains the culture of two partsCell.For example, an at least film can be removed, so that a part culture cell still adheres to, or removes or puncture to sample trainingSupport cell.In operating procedure 1314, a part culture cell is put to death for identifying.Step 1314 may include PCR, sequencingAnd/or various data analyses.In step 1316, aimed strain is identified.Such as aimed strain and/or remainder can be usedCulture cell is further cultivated, tested and/or is identified.Alternatively, being screened in step 1318 to chip application function.In step 1320, one or more variables are observed, aimed strain is identified in step 1316.
Figure 14 is the schematic diagram for illustrating the screening technique according to some embodiments.Small Figure 140 0 shows complex sampleExample, especially micropopulation sample 1402 and pedotheque 1404.In small Figure 140 6, using for example shown in Fig. 5 A and 5BScheme is from least one cell of sample extraction.In small Figure 140 8, cell (and any environmental extract that at least one is extractedAnd/or diluent) it is loaded to micromachining device or chip 1410 at least one high density microwell array.It can be by chip1410 and kit 1412 be put into couveuse 1414.Reagent can be used for adding liquid to maintain growth and/or various screening purposesNutritional need.Small Figure 141 6 shows output: the isolated bacterial strain of the selection result and culture cell.
Figure 15 is a series of image of explanations according to the screening example of some embodiments.Image show with film andApplication has a part of the chip of acid-sensitive layer thereon, to screen low pH.In image 1500, it is seen that micro- more than 1800 50 μmHole, 9 clear hits 1502.Image 1504 is the enlarged drawing of frame 1504, and image 1506 is a micropore with hit 1502Enlarged drawing.
Figure 16 A-16C is the image illustrated according to some embodiments from screening recycling.In Figure 16 A, microscope is usedAt least one hole is chosen with the selecting device at least one needle.In fig. 16b, needle is removed and is incubated in the medium.In Figure 16 C, it is seen that growth.
Figure 17 A is the exploded view for illustrating the chip for screening according to some embodiments.In Figure 17 A, chip1700 include the high density microwell array in micropore with such as edaphon.Film 1702 is applied to chip 1700.In filmDirection chip 1700 applies gasket 1704 on 1702.Direction chip, which is applied, on gasket 1704 and film 1702 has fluorescence large intestine barThe agar 1706 of bacterium.Figure 17 B and 17C are the images for illustrating the screening example according to some embodiments.In this example, it screensKeep-out region.Figure 17 B is the fluorescent image of chip after screening, and the chip is prepared as the chip 1700 in Figure 17 A.Figure 17 CIt is to show the image for the process for choosing sample by agar from the chip.
In some embodiments, the position in equipment may be moved with a part (or part of a part) sample from equipmentExisting a part of sample is related at this location after removing.Equipment can be microarray or including microarray.Microarray canTo include multiple positions for applying sample, wherein each position is marked with unique label, and the unique label is for identifyingA part of sample from microarray removal after, the sample segment from position.
This disclosure relates to for identifying that described a part contains at least one core in a part of sample after microarray removalThe method of which position of the sample of acid molecule on microarray, method includes the following steps: (a) is in the multiple of microarrayThe sample of one or more parts is applied in the one or more of position, wherein unique label of each position comprising nucleic acid moleculesLabel, the unique label includes: (i) location specific nucleotide sequence；(ii) first target specificity nucleotide sequence；(b) the target nucleic acids molecule found and the label of mark position in the sample of at least one portion is allowed to anneal；(c) to annealingNucleic acid molecules group carries out primer extend, reverse transcription, single-stranded connection or double-strand connection, so that location specific nucleotide sequence is wholeIt is incorporated into each nucleic acid molecules generated by primer extend, reverse transcription, single-stranded connection or double-strand connection；(d) merge in step (c)The nucleic acid molecules group of generation；(e) combined nucleic acid molecules group is sequenced, to obtain one or more location specific nucleotides sequencesThe sequence of column；(f) by least one the location specific nucleotide sequence obtained from combined nucleic acid molecules group with comprisingPosition on the microarray of the label label of the location specific nucleotide sequence is associated, so that identification includes at least oneWhich position of a part of sample of nucleic acid molecules from microarray.In some embodiments, sample may include at least oneA cell, and one or more nucleic acid molecules are discharged from cell after step (a) and before step (b).Sample may includeAt least one cell, and after the step (a) and at least one cellular replication of step (b) foregoing description or division.In step (b)Before, a part of a part of sample can be removed from least one position, and a part of of a part of sample can depositIt is stored in independent container relevant to the initial position of a part of sample described on microarray.The method of association or identification position canTo further include steps of the combined nucleic acid generated in the nucleic acid molecules generated in amplification step (c) or step (d) pointSubgroup.Amplification step may include polymerase chain reaction amplification, multiplex polymerase chain re-action amplification, nested polymerase chainReaction amplification, Ligase detection reaction amplification, strand displacement amplification, the amplification based on transcription, is based on ligase chain reaction amplificationAmplification, rolling circle amplification or the hyperbranched rolling circle amplification of nucleic acid sequence.Other primers can be added in the amplification reaction.For example, PCRReaction needs both 5' and 3' primers.A primer can be complementary with the nucleotide sequence in sample used in amplified reaction.
It in some embodiments, can include thin with nucleic acid enzymatic treatment before applying composition to micromachining deviceThe composition of born of the same parents and/or virus, so that contaminated nucleic acid molecule is not amplified in later step.
Used in disclosed method and apparatus sequencing can be obtain sequence information any method, including hybridization andIt uses sequence specific protein (such as enzyme).Sequencing may include Sanger sequencing, by sequencing by hybridization, by connection sequencing,Quantitative increment fluorescent nucleotide addition sequencing (QIFNAS), gradually connection and cutting, fluorescence resonance energy transfer, molecular beacon,The digestion of TaqMan reporter gene probe, pyrosequencing, fluorescent in situ sequencing (FISSEQ), wobble frequency sequencing, multiple sequencing, polymerization(see, for example, U.S. Patent Application Publication No. 2012/0270740, full text is incorporated by reference into this for bacterium colony (POLONY) sequencingText)；(see, for example, U.S. Patent Application Publication No. 2009/0018024, full text passes through for nanometer lattice rolling ring (ROLONY) sequencingBe incorporated herein by reference), the connection measurement sequencing of allele-specific oligomerization, or the sequencing on next-generation sequencing (NGS) platform.The non-limiting example of NGS platform includes coming from system below:(San Diego, California) (exampleSuch as, MiSeqTM/NextSeqTM/HiSeqTMWith HiSeq XTM)、Life Technologies(Carlsbad,California)(such as Ion TorrentTM), and Pacific Biosciences (Menlo Park, California) (such asRS II)。
Organism or type obtained from the nucleic acid sequence of the organism and can contain each of organism sequence by comparingDatabase is planted to identify.For example, ribosomal RNA sequences data can be from SILVA rRNA database project (Max PlanckInstitute for Marine Microbiology,Bremen,Germany(www.arb-silva.de)；See, for example,Quast etc., " The SILVA Ribosomal RNA Gene Database Project:Improved DataProcessing and Web-Based Tools, " 41Nucl.Acids Res.D590-D596 (2013), and PruesseDeng " SINA:Accurate High-Throughput Multiple Sequence Alignment of RibosomalRNA Genes, " 28Bioinformatics 1823-1829 (2012), the two full text are both incorporated herein by reference) obtain.Other ribosomal RNA sequences databases include ribosomes database project (Michigan State University, EastLansing,Michigan(www.rdp.cme.msu.edu)；See, for example, Cole etc., " Ribosomal DatabaseProject:Data and Tools for High Throughput rRNA Analysis"42Nucl.AcidsRes.D633-D642 (2014), is hereby incorporated herein by reference) and Greengenes (Lawrence BerkeleyNational Laboratory,Berkeley,California(www.greengenes.lbl.gov)；See, for example,DeSantis etc., " Greengenes, a Chimera-Checked 16S rRNA Gene Database and WorkbenchCompatible with ARB, " 72Appl.Environ.Microbiol.5069-72 (2006), full text is by quoting simultaneouslyEnter herein).Genetic sequence database includes publicly available almost 260,000 species formally describedNucleotide sequence (National Institutes of Health, Bethesda, Maryland(www.ncbi.nlm.nih.gov)；See, for example, Benson etc., " GenBank, " 41Nucl.Acids Res.D36-42(2013)。
Sequence for matching and identifying may include 16S ribose body region, 18S ribose body region or offer authentication informationAny other region.Required variant can be genotype (such as single nucleotide polymorphism (SNP) or other kinds of variant)Or the type comprising specific gene sequence (such as sequence of codase or protein).Organism or type can also by by itsSequence is identified with customization internal sequence database matching.In some cases, if obtained from a part of sample of some positionThe sequence obtained is with known dna, cDNA or the RNA sequence obtained from the type or microorganism with the same of at least particular percentileProperty (for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100% identity), it can be concluded that and find the type or micro- in the position of microarrayBiology.
The disclosure further to including method for the microarray for applying multiple positions of sample for manufacturing, whereinAt least one position is marked with unique label, the described method comprises the following steps: multiple labels (a) is synthesized, wherein each labelComprising nucleic acid molecules, the nucleic acid molecules include: (i) location specific nucleotide sequence；(ii) target specificity nucleotideSequence；(b) label is placed at least one position of multiple positions on microarray.In another embodiment, originallyOpen to be related to for manufacture including method for the microarray for applying multiple positions of sample, wherein at least one position is with solelySpecial label label, the described method comprises the following steps: (a) synthesizing multiple labels, wherein each label includes nucleic acid molecules, it is describedNucleic acid molecules include target specificity nucleotide sequence and do not include location specific nucleotide sequence；(b) label is placed inOn at least one position of multiple positions on microarray.Target specific sequence on each position of microarray can be identical.In any the embodiment above, step (a) can carry out before step (b).Placing step (b) may include passing through liquidLabel is placed in respectively by operation sequence (for example, liquid relief, with solid syringe needle point sample, with hollow pinhead point sample or with ink discharge device deposit)A position.At least one label may include a part of pre-synthesis nucleic acid molecules or nucleic acid molecules.Step (a) can be withStep (b) carries out simultaneously.In some embodiments, at least one label includes the nucleic acid molecules of synthesis, at various locationsPass through fabricated in situ.Synthesis step (a) may include inkjet printing synthesis or photoetching synthesis.
Each position on microarray can be set to receive a part of sample.Nucleic acid molecules (such as few nucleosides can be usedAcid) label or tag position, the nucleic acid molecules include at least one: (i) location specific nucleotide sequence (bar code)；With(ii) target specificity nucleotide sequence.Target specificity nucleotide sequence can be complementary with the nucleotide sequence found in sampleOr it is substantially complementary.The sequence of nucleotide sequence in nucleic acid molecules from the end 5' to the end 3' can be (i) location specificNucleotide sequence；(ii) target specificity nucleotide sequence.Alternatively, the nucleotide in nucleic acid molecules from the end 5' to the end 3'The sequence of sequence can be (2) then (1).Nucleic acid molecules can be connected to microarray in its end 5'.Equipment (such as micro- battle arrayColumn) on one or more positions can be without label or unmarked.
Term " complementation " or " being substantially complementary " can refer between nucleotide or nucleic acid, such as such as double chain DNA moleculeTwo chains between or Oligonucleolide primers and single-chain nucleic acid on primer binding site between hybridization, base pairing or formationDuplex.In general, complementary nucleotide is A and T/U or C and G.When optimal comparison and compare and have appropriate nucleotides inserted orMissing a chain nucleotide and another chain at least about 80%, typically at least about 90%-95%, more preferably from about 98-100% oligonucleotide ligand clock synchronization, two single stranded RNAs or DNA molecular are known as being substantially complementary.Alternatively, when RNA or DNA chain will beWhen hybridizing under selective cross condition with its complement, there are basic complementarity.In general, when in one section of at least 14-25 nucleosidesWhen there is at least about 65% complementarity, at least about 75% or at least about 90% complementarity on acid, selective cross will occur.
Term " selective cross " refers to detectable specific binding.Polynucleotides, oligonucleotides and its segment are bigAmount and the detectable combination of non-specific nucleic acid minimum hybridize and wash conditions under with nucleic acid chains selective cross.It can makeThe selective cross condition known in the art being discussed herein is realized with " high stringency " condition.The example of " high stringency " condition isA kind of method for being incubated for another polynucleotides with polynucleotides, one of polynucleotides can at 42 DEG C of hybridization temperatureHybridization buffer (the piece that 6x SSPE or SSC, 50% formamide, 5x Denhardt reagent, 0.5%SDS, 100 μ g/ml are denaturalizedSectionization salmon sperm dna) in be fixed to the surface of solids (such as film) 12-16 hours, then with washing buffer (1x SSC,0.5%SDS) washed twice at 55 DEG C.
Nucleic acid molecules as location tags a part may include at least one deoxyribonucleotide or at least oneRibonucleotide.Nucleic acid molecules can be single-stranded or double-stranded.Nucleic acid molecules can be the duplex molecule with single-stranded outstanding end.
In some embodiments, location tags can be used for expanding the nucleic acid molecules annealed with it.Therefore, location tags canTo include the nucleic acid sequence further containing amplimer binding site.The length of amplimer binding site can be at least 16It is a, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, extremelyFew 25, at least 26, at least 27, at least 28, at least 29 or at least 30 nucleotide.From the end 5' in nucleic acid moleculesSequence to the nucleotide sequence of the end 3' can be for example: (1) amplimer binding site；(2) location specific nucleotideSequence；(3) target specificity nucleotide sequence.
In some embodiments, nucleic acid molecules may include target specificity nucleotide sequence and not include position specialProperty nucleotide sequence.In certain embodiments, nucleic acid molecules may include target specificity nucleotide sequence and not include positionSet specific nucleotide sequences or amplification binding site sequence.In a further embodiment, nucleic acid molecules can only includeTarget specificity nucleotide sequence.In even further embodiment, nucleic acid molecules can only include target specificity coreNucleotide sequence.Amplimer binding site can combine polymerase chain reaction primer, multiplex polymerase chain re-action primer, nestFormula polymerase chain reaction primer, Ligase detection reaction primer, strand displacement primer, is based on turning ligase chain reaction primerThe primer of record, the primer based on nucleic acid sequence, rolling ring primer or hyperbranched rolling ring primer.It can be to microarray during amplified reactionOther primers are added.For example, PCR reaction needs both 5' and 3' primers.Target specificity nucleotide sequence can contain targetThe position amplification of nucleic acid molecules is marked, and can be come for example, by the dyestuff of qPCR, terminal PCR and/or detection amplifier nucleic acid moleculeDetection.
It is expected that the nucleic acid molecules annealed with location tags or amplified production based on this nucleic acid molecules are sequenced.PositionSetting label may include nucleic acid sequence further containing aptamer nucleotide sequence.In certain embodiments, it is adapted to daughter nucleusNucleotide sequence is not present in location tags, but is added into secondary PCR reaction or through connection to sample nucleic acid molecule.It is suitableGamete nucleotide sequence can be General adaptive or specific microarray dataset (such asOr Ion TorrentTM)Aptamer.Aptamer nucleotide sequence may include sequencing primer binding site.Sequencing primer binding site can be combined and is used forPrimer below: Sanger sequencing is sequenced, quantitative increment fluorescent nucleotide addition sequencing by sequencing by hybridization, by connection(QIFNAS), gradually connection and cutting, fluorescence resonance energy transfer, molecular beacon, TaqMan reporter gene probe digestion, coke phosphorusSour sequencing, the sequencing of fluorescent in situ sequencing (FISSEQ), wobble frequency, multiple sequencing, polymerization bacterium colony (POLONY) sequencing are (see, for example, beautyState's patent application publication number 2012/0270740, is hereby incorporated herein by reference)；Nanometer lattice rolling ring (ROLONY) is sequenced(see, for example, U.S. Patent Application Publication No. 2009/0018024, being hereby incorporated herein by reference), allele specificProperty oligomerization connection measurement sequencing, the sequencing on NGS platform or any suitable sequencing program.The non-limiting example of NGS platformIncluding coming from system below:(San Diego, California) (such as MiSeqTM/NextSeqTM/HiSeqTMWith HiSeq XTM), Life Technologies (Carlsbad, California) (such as Ion TorrentTM),With Pacific Biosciences (Menlo Park, California) (such asRS II)。
The length of location specific nucleotide sequence (such as bar code) is at least two, at least three, at least four, at least 5A, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14It is a, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, extremelyFew 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or at least 30 nucleotide.
The length of target specificity nucleotide sequence be at least ten, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22,At least 23, at least 24, at least 25 nucleotide, at least 26, at least 27, at least 28, at least 29, at least 30A, at least 40, at least 50, at least 75 or at least 100 nucleotide.
At least one position on microarray can further be marked with unique molecular identifier tags.Unique molecular markSymbol can be used for quantitative growth (such as the growth of microbe colony or cellular replication in the position).Unique molecular identifier can beRandom nucleotides.The reality of method and unique molecular identifier using unique molecular identifier has been described in the prior artExample, the WO 2013/173394 being hereby incorporated herein by reference see, for example, it.For example, the core of unique molecular identifier tagsNucleotide sequence can be NNNANNNCNNNTNNNGNNNANNNCNNN (SEQ ID NO:1), and wherein (equivalent of ACGT is random by NsMixing) big coding space is generated, so that it (is 4 in the example that each molecule of amplification, which obtains unique (specific) DNA sequence dna bar code,^N bar code, or 4^21~40 are hundred million).The sequence can count, without by the interference from amplification preference or other technologies problem.Fixation base (A, C, G, T) in SEQ ID NO:1 helps accurately to read bar code, such as processing indel.
The disclosure, which covers using location specific label, monitors more than one target specificity nucleotide sequence in samplePresence or content (for example, multiple).At least one position on microarray can be further with only comprising nucleic acid molecules secondSpecial label label, the nucleic acid molecules are including, for example, (i) amplimer binding site: (ii) location specific nucleotide sequence；(iii) target specificity nucleotide sequence.
In some embodiments, nucleic acid molecules may include target specificity nucleotide sequence and not include position specialProperty nucleotide sequence.In certain embodiments, nucleic acid molecules may include target specificity nucleotide sequence and not include positionSet specific nucleotide sequences or amplification binding site sequence.In a further embodiment, nucleic acid molecules can only includeTarget specificity nucleotide sequence.In even further embodiment, nucleic acid molecules can only include target specificity coreNucleotide sequence.The length of target specificity nucleotide sequence can at least ten, at least 11, at least 12, at least 13,At least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25 nucleotide, at least 26, at least 27, at least 28, at least 29, extremelyFew 30, at least 40, at least 50, at least 75 or at least 100 nucleotide.In certain embodiments, in microarrayThe target specific sequence of each position can be identical.Other target specificity nucleotide sequences can be monitored.For example, can useAt least ten, at least 25, at least 50, at least 75 or at least 100 unique labels mark one or more positions, whereinEach label includes the target specificity nucleotide sequence different from other target specificity nucleotide sequences in the location tags.
Any target gene position can provide target specificity nucleotide sequence.Such as following sequence: bacterial 16 S riboseBody RNA (rRNA), 18S rRNA, poly- (A) RNA, rna polymerase gene, DNA polymerase gene, RecA gene, transposaseGene, ribosome internal transcribed spacer (ITS) sequence, the gene of codase, control zone DNA sequence dna, binding site DNA sequenceColumn, or can be used as a part of any of these sequences of target specificity nucleotide sequence.Disclosed system, is set kitThe one or more bacterial 16 S rRNA primers described in the following: Sundquist etc. can be used in standby or method, "Bacterial Flora-Typing with Targeted,Chip-Based Pyrosequencing,"7:108BMCMicrobiology (2007) and Wang etc., " Conservative Fragments in Bacterial 16S rRNAGenes and Primer Design for 16S Ribosomal DNA Amplicons in MetagenomicStudies, " 4:10PLoS ONE e7401 (2009), is hereby incorporated herein by reference.
The sample used in disclosed device and method may include multiple nucleic acid molecules.Sample may include at least oneA DNA molecular or at least one RNA molecule.Sample may include at least one nucleic acid molecules formed by restriction Enzyme digestion.Sample may include at least one cell (such as archeabacterial cell, eubacteria cell, fungal cell, plant cell and/or animalCell).Sample may include at least one microorganism.Sample may include one or more viral (such as bacteriophages), to thisIt may need to provide host cell.A part of sample of a position can be individual cells or raw from individual cells on microarrayLong bacterium colony.For example, individual microorganism or cell can be placed in micropore and allow the individual microorganism or cellDivision or duplication so that bacterium colony is grown in each micropore, and wherein have individual microorganism or cell.Therefore, on microarrayPosition may include single microbial type or the growth that supports one another microbial strains mictium.Sample may includeAny suitable diluent.In non-limiting example, sample includes soil, sewage, fecal matter, body cavity content, biology streamBody, living organism, dead organic matter, microbial suspension, natural fresh water, drinking water, seawater, waste water, overcritical titanium dioxideCarbon, mineral, gas, buffer, alcohol, organic solvent and/or oil.In some embodiments, it is placed in by a part of sampleBefore at least one position of microarray, the position will be placed in comprising nucleic acid molecules below: (a) (i) location specific nucleotideSequence and (ii) one or more target specificity nucleotide sequences；Or (b) one or more target specificity nucleotide sequences(that is, not including location specific nucleotide sequence).In other embodiments, a part of sample is being placed in microarray extremelyBehind a few position, the position will be placed in comprising nucleic acid molecules below: (a) (i) location specific nucleotide sequence and (ii)One or more target specificity nucleotide sequences；Or (b) one or more target specificity nucleotide sequences (that is, not includingLocation specific nucleotide sequence).In an example, sample or a part of sample are placed on microarray and are incubated for, thenTo be placed on microarray comprising at least one nucleic acid molecules below: (i) location specific nucleotide sequence and (ii) target are specialSpecific nucleotide sequence.In some embodiments, by a part of a part of sample from least one on microarrayPosition remove, and store in a separate container, or before nucleic acid molecules to be placed in at least one position on microarray orMicroarray is separated later.
At least one location specific label may include pre-synthesis and be placed in the position by liquid operation sequenceA part of nucleic acid molecules or nucleic acid molecules.For example, liquid operation sequence can be liquid relief, with solid syringe needle point sample, with hollowSyringe needle point sample is deposited with ink discharge device.Multiple nucleic acid molecules separately synthesized in advance can be used and generate label in position.ExtremelyA few label may include to be synthesized by fabricated in situ (such as being synthesized by inkjet printing synthesis or photoetching) in the positionNucleic acid molecules.
The digital counting of type
This document describes the superchip devices comprising the surface with high density micropore.It can will come from micropopulationThe microorganism dilution of sample is simultaneously applied to device, so that hole includes about microorganism/occupy hole.Then, chip is incubated for makeMicroorganism is replicated in hole.Further, this document describes the location index systems based on DNA, and to measure, there are what in each holeType.The directory system may relate to for PCR primer to be loaded to each hole in advance, and the PCR primer contains the addressing item in identification holeThe offer information of specific gene sequence (such as 16S) in code and targeted microorganisms genome targets required gene orderPrimer sequence.After incubation, releasing microbe DNA, PCR primer expands non-target bacteria region of DNA domain, and collects each on chipThen the amplicon in a hole is read by next-generation sequencing.
System as described herein, kit, device and method can be used for carrying out each microbe species in sample or variantThe absolute counting of quantity.Each hole can represent a digital event, and single microbial deposits in expression original dilution sample?.It is any bacterial species that location index system, which allows user to measure in hole,.Measurement unit can be that " there are bacterium kinds in holeClass ", and can be unrelated with the quantity of bacterium in hole.
In an example, the mixing sample of microorganism includes the type of the type 2 and 20% of 50% type 1,30%3.Then dilute sample is applied to chip, so that the hole (major part) respectively occupied has a kind of microorganism.Microorganism duplication.NoteMeaning, different types of reproduction speed may be different.Then, chip is processed, so that the DNA in hole from microorganism is discharged, and16S or some other target sequences are expanded.The DNA cloning product from each hole can be collected, and is carried out with next generation's sequencingSequencing.Next-generation sequencing data be can analyze to measure for each hole, be what type respectively occupying in hole.Many holes canIt can not be occupied.Can pass through the various types of abundance of following measurement: the sum in the various types of hole occupied is divided by occupying holeSum.Following measurement absolute abundance can be passed through: by the various types of abundance % from step multiplied by microorganism in primary sampleSum.Sequencing data can be compared with publicly available sequence database, what type is respectively occupied in hole with measurement is.For example, ribosomal RNA sequences data can be obtained from above-mentioned SILVA rRNA database.Other ribosomal RNA sequences databases are alsoIncluding above-mentioned ribosomes database project, Greengenes andGenetic sequence database.
Method currently used for the abundance of microbe species in estimation sample is related to using traditional technology, such as microscope,Dyeing, selective medium, metabolism/physiology screening and with culture dish culture.These methods are typically due to lack specific (micro-Mirror, dyeing, selective medium) or lack the ability (selective medium, culture) of all kinds in count samples without essenceReally, and many types grown with conventional method it is bad or do not grow.
Molecular method currently used for microbe species relative abundance in measurement micropopulation sample is related to from sample extractionMicrobial DNA carries out PCR amplification to the 16S or some other region of DNA domains that provide type or other information, then to gained PCRProduct carries out next-generation sequencing (NGS).Speculate in primary sample from the relative frequency of species specificity DNA sequence dna in NGS dataVarious types of relative abundance.For document about many examples of this alanysis, this method is that base is laid in the research of many micropopulationsPlinth.
It is following the problem of method to be at present that it is not controlled: the quantity for being present in the 16S gene in different microorganisms may notTogether, PCR preference (wherein the sequence of different microorganisms type may be expanded with different rates) and sequencing preference (wherein different micro- lifesThe sequence of species may be sequenced with different rates).As a result, the accuracy of the relative abundance data obtained with current methodThere are many uncertainties.
Different types of counting can be based on the presence of type in single hole.This be divided into during loading in hole from originalThe single microbial of beginning sample is directly related.Only PCR/NGS can be used for identifying that there are what microorganisms in each hole.The sequence of identificationThe quantity of column is not from a part of calculating.Therefore, PCR, NGS or the variation of target sequence copy number or preference do not weigh in methodIt wants.
Some embodiments can have below application: micropopulation research, microniological proudcts are developed and are developed, clinicalDiagnosis and any other field for wherein needing the microbe species in accurate metering sample.
Therefore, some embodiments can provide the measurement that various types of relative abundance is much more accurate in micropopulation sample withAnd by the relative abundance measurement to convert the various types of ability directly counted in absolute abundance or primary sample (dilute by calculatingRelease and/or in conjunction with microbial count in primary sample measurement).Some embodiments can provide for high density micro Process chipNew opplication (in addition to cultivating and screening microorganism).
Figure 18 is the flow chart for illustrating the method for counting according to some embodiments.In step 1800, sample is obtained.?In step 1802, from least one cell of gained sample extraction.In step 1804, at least the one of micromachining device or chipThe cell of at least one extraction of loading in a high density microwell array.Step 1804 may include the cell extracted at least oneIt prepares cell concentration object, select at least one nutrients/culture medium, and/or a selection at least film.In step 1806, useThe film of at least one selection seals at least part of microwell array so that cell concentration object to be retained in micropore.In step 1808In, it is incubated for chip.Step 1808 may include selection temperature, determine atmosphere (such as aerobic or anaerobism) and/or incubation time.In step 1810, a part culture cell is put to death for identifying.Step 1810 may include PCR, sequencing and/or various dataAnalysis.In step 1812, it can be estimated that and/or information (for example, biological community structure) of the measurement about sample.
Figure 19 is the schematic diagram for illustrating the method for counting according to some embodiments.Small Figure 190 0 shows complex sampleExample, especially micropopulation sample 1902 and pedotheque 1904.In small Figure 190 6, using for example shown in Fig. 5 A and 5BScheme is from least one cell of sample extraction.In small Figure 190 8, at least one cell extracted (and any environmental extractAnd/or diluent) it is loaded to micromachining device or chip 1910 at least one high density microwell array.It can be by chip1910 and kit 1912 be put into couveuse 1914.Reagent can be used for adding liquid to maintain growth and/or various screening purposesNutritional need.Small Figure 191 6 shows output: the relative abundance of sequence and culture cell.
Platform based on drop
Platform based on discrete droplets can be used for separating, cultivate and/or screen, with the basic phase of mode for using chipTogether.Drop is micropore analog, is used as nanometer or picoliters container.Drop forming method, especially when with Cell Sorter on chipWhen type instrument combination, it can be used for isolating microorganism from complex environment sample.Drop addition can be used for feeding microorganism.Drop pointIt splits and can be used for sequencing or other destructive testings, while leaving sample living.All preparations needed for sequencing can also be with dropForm carries out.
Some embodiments can be used for taking out microorganism from complex environment, become drop.For example, be used to prepare containingThe regulator control system of the drop of cell suspending liquid may include one or a small amount of cell.It can be by water drop suspension in unmixing liquidBody makes them keep separation each other, and prevents from contacting or polluting any surface.It can be generated in each microchannel in such as 30HzDrop is converted into millions of daily.
Microfluidic system based on drop can encapsulate, manipulate and/or be incubated for droplet (for example, about 30pL).It noticesCell survival and proliferation are similar with the control experiment in bulk solution.Drop can be generated in hundreds of Hz, it is meant that can be severalMillions of drops are generated in a hour.The simply device based on chip can be used for generating drop, and drop can be engineered withInclude individual cells.
Some embodiments can be used for screening the cell in drop.It can be with 500 drops for example per second on chipDrop after the incubation of rate fluorescent screening.Drop, which can flow through, can be set to show for the epi-fluorescence of a variety of different measurementsThe channel of the focal point of micro mirror.This may be the screening particularly effective method of metabolin, because due to being confined to very small liquidDrop, so local concentration is quite high.
Some embodiments can be used for sorting drop.Once cell can sort them by separation, growth and/or screeningSo that obtaining useful sample.Can by with as common FACS machine type in a manner of drop is sorted.
Some embodiments can be used for dividing drop.Some embodiments may need the energy for obtaining sample and being dividedPower so that a part is sent to sequencing (disruptive method), and is retained as another part of culture living.There are many points at presentThe distinct methods of drop are split, is including but not limited to intersected with the size configurations T shape carefully calculated, is caused in flowing or electrowettingIt generates drop breakup (being careful not to cause cell cracking with too high voltage).
Some embodiments can be used for merging drop and/or add reagent to drop.For example, the long-time of cell is incubated for(such as a few weeks) needs the ability of adding liquid, to maintain the nutritional need of growth.Reagent is added for various screening purposesIt is useful.Drop screening is dependent on the drop containing compound password and capable of containing the droplet coalescence of individual cells.SoAfter can be incubated for drop, and/or return it into analysis chip with by its password authentication compound.This may be needed as neededThe ability of precise merging drop.
Some embodiments can be used for carrying out PCR in drop.PCR can be used for finally being sequenced specific genetic elements (such asThe region 16S), to identify microorganism.It is what kind of microorganism that this, which can be used to measure grow in each hole,.It is being based onIn the system of drop, it is any microorganism present in each drop that this method, which can be used for measuring, as long as devising amplification gene groupThe correct primer sequence in correct region.
Some embodiments can be used for being sequenced from drop DNA (such as generating in a PCR step), and/or preparation DNA textLibrary.
Location specific label for superchip
The superchip device on the surface with high density micropore can be used.It can will be from micropopulation sampleMicroorganism dilution is simultaneously applied to device, so that hole includes about microorganism/occupy hole.Chip, which can be incubated for, makes microorganism existIt is replicated in hole, gained group represents single type.Location index system based on DNA can be used for measuring in each hole there are what plantClass.The directory system may relate to for PCR primer to be loaded to each hole in advance, and the PCR primer contains the addressing bar code in identification holeWith the primer sequence (such as 16S of microbial genome) for the specific genetic elements of targeting for providing information.After incubation, releaseMicrobial DNA, PCR primer expand non-target bacteria region of DNA domain, the amplicon in each hole on chip are collected, under then passing throughGeneration sequencing is read.
Above-mentioned location index system may relate to each hole that different location password is added to microchip, or by multiple positionsEach hole is added in password, so that password sum needed for encoding certain amount of hole is reduced.For example, as having 100 on fruit chipHole, if one, each hole password, needs 100 passwords.If reading two passwords from each hole, same chip can be onlyWith 20 cipher codings (that is, the x-axis of 10 coding grids, 10 coding y-axis).
Table 2 provides the example that the PCR strategy of two passwords is added in each hole.
Table 2
Table 3 provides the example that the PCR strategy of three passwords is added in each hole.
Table 3
Three oligomer primers are for manufacturing single PCR product.The system is put using two oligonucleotides in one end of moleculeThe advantages of setting multiple slitting codes may include the maximum length or manufacture overlength bar code of oligonucleotides needed for for example reducing.
This method may be summarized to be each reaction and n bar code be added.This method can also have different implementations, so that bar code existsThe same side in target sequence region.NGS sequencing aptamer can be used, and read bar code PCR product group using next-generation sequencingFull sequence.
In another kind is implemented, fixed bar code can be added to indicate sample size or plate quantity, and allow in two itemsCollect multiple sample/plates in the operation of code, as shown in table 4.
Table 4
In another kind is implemented, fixed bar code can be added to indicate sample size or plate quantity, and allow in three itemsCollect multiple sample/plates in the operation of code, as shown in table 5.
Table 5
Note that in all cases, information, therefore CODE1, CODE2 and CODE3 item are conveyed in the position of bar code in the sequenceCode is not necessarily different from each other in particular bore.
Prepare oligonucleotide and with solid size coded system printing chip higher cost.For example, the chip in 10,000 holesIn the hole for needing 10,000 single bar codes and 10,000 independent print cycles so that these bar codes to be placed on chip.If madeWith dicode system, only 200 bar codes and 200 print cycles may be needed to manufacture chip.This means that substantial saved widowNucleotide cost, print time and printing capital input.
Using dual bar code PCR primer, amplification and sequencing analysis are then carried out, is assigned randomly to micro Process chip to provideOn DNA or the position data of the part containing DNA may have very high practicability and relatively low cost.
Figure 20 is the schematic diagram for illustrating the directory system according to some embodiments.Microwell chips 2000 have N row and MColumn, to generate NxM unique index.It is considered that the position of micropore has coordinate (N, M) in chip 2000.Each column has normalReverse primer sequences (for example, R1, R2, R3..RM) and every row have common forward primer sequence (for example, F1, F2,F3..FN).For example, the unique label of specific genetic elements may include just in target such as 16S ribosomes nucleotide (rRNA)To primer sequence F515 and reverse primer sequences R806.It, can be based on forward primer sequence and anti-after chip 2000 is carried out PCRThe presence of target genetic elements is mapped back unique original micropores by the presence to primer sequence.For example, F515's and R806 depositsIn the micropore that will there are coordinate (515,806) in user guiding chip 2000.
The variability of pcr amplification product containing germy micropore reduces
The location index system based on DNA can be used, and to measure, there are what types in each hole.The directory system mayIt is related to for PCR primer being loaded to each hole in advance, the PCR primer contains the addressing bar code and targeted microorganisms genome in identification holeSpecific genetic elements (such as 16S) offer information primer sequence.After incubation, releasing microbe DNA, PCR primerNon-target bacteria region of DNA domain is expanded, the amplicon in each hole on chip is collected, is then read by next-generation sequencing.
Some embodiments of the variation of the amount of PCR product may include the limiting holes during manufacturing chip between limiting holesThe amount of middle PCR primer, so that the amount of PCR primer is in DNA amplification reaction for most of possible sample DNA concentrationIt is limited, so that difference of the amount of the PCR product generated between each hole will be smaller.
Some embodiments of the variation of the amount of PCR product may include by the recurring number of PCR on chip between limiting holesIt is limited to less than 3 circulations or less than 5 circulations or less than 10 circulations or less than 15 circulations or less than 20 circulations,Or less than 25 circulation or less than 30 circulation so that relative to complete alternation PCR amplification scheme, the amount of the PCR product of generation existsDifference between each hole will be smaller.
Some embodiments of the variation of the amount of PCR product may include the core in limited reactions mixture between limiting holesThe amount of thuja acid, so that the quantity of PCR amplification of generation and the amount of nucleotide are more relevant compared with the amount of DNA in primary sample.Micropore with a large amount of target DNA will exhaust nucleotide in cyclic process early stage, and the micropore with a small amount of target DNA will followThe ring process later period exhausts nucleotide, but generates the amplified production of nearly identical amounts.
Some embodiments of the variation of the amount of PCR product may include being limited in the micro- life grown in hole between limiting holesThe amount of the available nutrients of object, so that cellular replication, until exhausting culture medium and then stopping replicating.
Some embodiments of the variation of the amount of PCR product may include that identification PCR is put into each hole between limiting holesThe dyestuff of product, so that the PCR product generated is more, signal is brighter.It can monitor the intensity of dyestuff during each PCR cycle, oneDenier observes required signal strength, and sample is taken out from hole.
Some embodiments of the variation of the amount of PCR product may include the mixture using hybridized bead between limiting holesWith with the DNA amplification selective cross from each hole, the hybridized bead is covered with the few core complementary with each hole specificity bar codeThuja acid.Once globule is saturated, uncombinating DNA is washed off, combines DNA from globule release.It then can be by the amount normalizing of the DNA in each holeChange to the saturation limit of globule.
Some embodiments of the variation of the amount of PCR product may include being incubated for chip for a long time between limiting holes, makeFast microorganism, which must be grown, could quickly fill up hole and stops replicating, and grows slow microorganism and gradually fills up hole, once and have in holeThe cell of about the same quantity then stops growing.
It in some embodiments, can be with using bar code primer and next-generation sequencing (NGS) in chip format and methodFor identifying which type is grown in which hole on high density microchip.When the bacterium of approximately equivalent quantity occupies on chipEach micropore when, the signal from each hole is about the same in NGS data.
For example, being generated at one in the typical NGS operation of 12,000,000 sequence reads, if 24 cores are sequenced in the operationPiece, every has bacterial number in 10,000 micropore and each hole identical, then 50 reads of average individual hole.
However, the different bacterium speed of growth is different, therefore a some holes may have seldom bacterium, and a some holes has veryMore bacteriums.This may make the operation of NGS become bad, because not detecting the seldom hole of bacterium in NGS analysis.
Therefore, in the example of typical NGS operation that 12,000,000 sequence reads are generated at one, each run is sequenced 24Chip, every have 10,000 micropore, more 100 times than the other half of the bacterium that wherein half has.It grows thin in slow holeThe probability that bacterium is detected significantly reduces.In this case:
(10,000x24)/2x100=12,000,000 (1)
(10,000x24) 72=120,000 (2)
Low frequency hole accounts for the 1% of sum.Therefore, in 12,000,000 read of NGS operation, 120,000Low frequency hole, i.e. 1 read of average individual hole will be come from.
To minimize this phenomenon, need to develop new method to keep the amount of the PCR product between hole equal, thusNGS operation detects all holes.
Silicon substrate microwell chips for microorganism separation, growth, screening and analysis
It substitutes or in addition to plastics, glass and/or polymer, micromachining device or chip can be by least part silicon structureAt to allow electrical measurement on the basis of hole-specifically.For example, the wall in each hole can be separated to generate micro- capacitor.At anotherIn example, FET makes a surface be exposed to the content in hole in each hole.Instead of pure silicon base chip, plating, gas can be passed throughMutually deposition, and/or electric arc/flame-spraying thin metal layer of upper generation at the top of existing chip.This can be more for chip additionFunctionality, using cheaper and/or more clean another manufacturing method, and/or allow hand-held or portable device smallType.
Some embodiments, which can permit to monitor by electrical measurement, to be grown.Microorganism can be measured using impedance monitoringThe growth of (such as bacterium).For example, in the Ur etc. that it is hereby incorporated herein by reference, " Impedance Monitoring ofBacterial Activity, " in 8:1J.Med.Microbiology 19-28 (1975), by the test tube containing Escherichia coliImpedance compared with its cell count.It can also be to other kinds of bacterium (including pseudomonas, Klebsiella and chainCoccus) to measure with proving effect be universal.Hole can be filled with different culture medium to test the growth of different preparationsCondition.
Some embodiments can permit is screened by electrical measurement.Electrical measurement can be carried out on the basis of hole-specifically,To be screened.One example will be pH.PH dependence is obtained on the door of mono- equipment of Kong Zhongcong there are many different modesReaction, including but not limited to ISFET and pH meter.It includes to produce that hole array with embedded pH sensor can measure those with electricityThe hole of the microorganism of raw acid or alkaline metabolin.One simple example is the generation from lactose screening lactic acid.Bacterium is existedIt dilutes, grow in hole, be then fed with lactose.Record pH decline hole contain can by lactose metabolism be lactic acid microorganism.
Some embodiments can permit electrical measurement redox probe.Another kind is to see using the method for electrical measurementExamine how the bacterium in hole influences known redox probe.Substantially, measurement there can be clear reaction in the presence of bacteriumSystem, and the deviation of expected property can be attributed to bacteria samples.Typical redox probe is similar to the iron cyanide: [Fe(CN)6]3-/4-.The iron cyanide is reduced to ferrocyanide by detailed characterizations, so that qualitative small variation, especiallyThe electronics transfer of surrounding them can be found.The system is " no label ", because it does not need to be directly changed bacterium itselfIt can be detected.
It can will identify that the antibody of microorganism (such as Escherichia coli) is fixed in ITO electrode.It can be surveyed from electrodeMeasure the resistance of the electronics transfer of the solution containing the iron cyanide.Escherichia coli are bound to electrode surface, with Escherichia coli on surfaceConcentration proportionally increase resistance.This is that redox probe can be used to detect certain types of microorganism or metabolinA kind of measurement method an example.
Work provides the advantage that including but not limited in silicon (or at least metal or metal plastic): production chip is more justPreferably (for example, by improving the prior art)；Allow small-sized and/or portable versions comprehensive detection abilities；In other materials (exampleSuch as LCR, CV) in the additional measurement capability that does not have；The newfound detection method based on chip is integrated in existing apparatus；And the combination with electrical measurement and sequencing.These advantages are beneficial for any consumer for using Interference Detection.
Protect the releasable barrier of the hole specific chemical object on chip
Figure 21 A-21E is the schematic diagram for illustrating the chip for having hole specific chemical object according to some embodiments.Figure 21 AShow micromachining device or chip with multiple micropores.In Figure 21 B, it is special in each micropore of chip to joined microporeProperty chemicals.In Figure 21 C, sealant is applied on the micropore specific chemical object in each hole of chip, to prevent chemicalsIt interacts with other additives in hole.In Figure 21 D, sample is added, sample is tested in micropore.In Figure 21 EIn, triggering agent (such as hot) discharges micropore specific chemical object, interacts with the sample in hole.
Microwell chips can be manufactured or be cleaned, and/or handle its surface.Specific chemical object can be prepared separately, thenSuch as using allowing to import a group-specific chemicals in specific one or the method and/or device adding hole in multiple holes.Then can be with application sealant to protect various chemicals not by environmental pollution, and/or it will with some specific external trigger agentIts removal/release/granting.
It can be particular design by micromachining device or chip manufacturing, such as clean and/or surface treatment to promoteWetting.PCR primer can be printed or pin mark enters particular bore.It is dry to can permit chip, then by evaporating from ethanol solutionDeposit wax layer.Optium concentration is about 1%v/v.Molten wax, or spraying aqueous or alcohol wax solution can directly be applied.Alternatively, can be withUse spin coating or vapor deposition.Various waxes can be used, including but not limited to: containing and the glycerol stearate without polyethylene glycolEster, cetostearyl alcohol, Cetyl OH, tristerin, glycerin monostearate, (the CAS registration of ceteareth -10Number 68439-49-6) and some commercial products, including but not limited to LotionproTM165 (can be fromEastsound, Washington are obtained) and PolawaxTM(can be obtained from Croda, Inc., Edison, New Jersey).ItAfterwards for example, by heating until wax thawing discharges following chemicals.It will be 50 DEG C -70 DEG C for these compositions.It is importantBe its sufficiently low aqueous solution without damaging any chemical component or boiling us.
Key concept is hole specific chemical object, is isolated on chip and discharges until experimenter triggers.This method canWith the coding for hole, but it can also be more broadly used for a series of different problems.It can be used for not assimilating for encapsulating chipLearn the various metabolism that object includes but is not limited to antibiotic, fluorescein, dyestuff, PCR primer, dissolution accelerator, antibody and/or testProduct.Although wax is to seal the good method of thing and can be discharged later by heating, other materials sealing can be used, work asIt is released when being exposed to light, ultrasound and/or some other triggering agent.Reagent is simply added compared to chip, this methodAdvantage first is that based on the control on hole-specifically (well-by-well).By printing chemicals into hole after progress Experiment on MicrobiologyIn may be implemented similar effect, but this can lead to the problem of (printing operation can be up to one day) with the time, in the known integrated circuit it is a fact that, ifMicroorganism on chip after fill each hole respectively, each hole can not at the same time in exposure.Using releasing mechanism, oftenIt a hole can be in same time exposure.In an example, wax can be deposited by solvent cast.
Simple and/or control relative abundance separation micropore
Superchip device may include the surface with high density micropore.It can will be from the micro- of micropopulation sampleBiology or other cell types dilutes and are applied to device so that hole comprising about microorganism or cell/occupy hole.It can useSemi-permeable film seals these micropores, and the semi-permeable film allows nutrients to diffuse into micropore, but prevents whole or at least oneBiology or cell are moved from micropore slightly.
Microorganism or cell sample can be prepared, then with impermeable or only gas-permeable film be sealed against intoChip.Only have the nutrients in hole to can be used for supporting micro- life in micropore when there is no liquid reservoir on chip or film, therefore sealingThe growth of object or cell.Two reasons of this feature include: that (1) building and workflow are simple, because device does not need half infiltrationPermeable membrane or storage cavern do not need addition nutrients, and possibility of pollution is smaller；(2) by limitation fast-growing type obtain nutrients comeCheck its relative abundance.For the sample comprising some fast-growing types and some slow raw types, fast-growing type will very in its respectively microporeIt fastly by limitation resource, stops growing or slow growth, while slow raw type continued growth.With wherein fast-growing type not by limitation nutritionThe case where amount of object, is compared, this makes slow raw type account for higher relative abundance in the microbiologic population on chip.When to chipOn all when being sequenced, this is extremely important for downstream processing.This also provides better detectable limit for rare type, becauseIt will not be surmounted by the fast type of growth to put rare type at some, to limit their ability of system detection.
Current method attempts that all kinds is allowed to grow, no matter they substantially grow it is fast or slow.It is inevitable in this wayAs a result, the leading group of fast-growing type and only relative abundance are increase with time.The downstream analysis of many types, such as sequencing or fluorescenceScreening can not differentiate each type in given group, but be only capable of differentiating and be higher than those of a certain limitation relative abundance.IfTarget is to retain diversity and detect rare type, needs to limit fast-growing type in some way.
For example illustrate the idea, consider a simple situation, sample includes two types: it is a kind of double daily,It is another double weekly, as shown in table 6.If raw type is rare (relative abundance 5%) when starting slowly, with two kindsThe growth of class, it becomes very rare quickly.
Table 6
It is slow raw after a period of time if limiting fast-growing type by the nutrition of growth and/or the competition of physical spaceThe relative abundance of type will start to increase, as shown in table 7.
Table 7
| Limitation | 0th day | 1st day | 2nd day | 3rd day | 7th day | 14th day |
| Fast-growing type | 19 | 38 | 50 | 50 | 50 | 50 |
| Slow raw type | 1 | 1 | 1 | 1 | 2 | 3 |
| It amounts to | 20 | 39 | 51 | 51 | 52 | 53 |
| The relative abundance of fast-growing type | 0.950 | 0.974 | 0.980 | 0.980 | 0.962 | 0.943 |
| The relative abundance of slow raw type | 0.050 | 0.026 | 0.020 | 0.020 | 0.038 | 0.057 |
High density micro Process array for biological library cell
Biological library is designed so that researcher obtains the great amount of samples from major community, to carry out certain form of grindStudy carefully, such as biomarker relevant to disease discovery.The sample that the prior art of biological storage art provides is stored in test tubeOr in low-density plank format, such as 96 holes or 384 orifice plates.When sample size to be stored is relatively fewer and sample itself be fromIt is useful in this way when scattered separation group.When storing following sample (such as micropopulation sample): wherein sample size mayThe quantity of variety classes or variant may be from each sample number hundred to thousands of or millions of a, current lifes in higher and each sampleObject storage method becomes very cumbersome.Use current method, it is necessary to carry out laborious separation scheme, so as to storing step itIt is preceding or isolate single type or variant later, to obtain desired type or variant.
System as described herein, kit, device and method can be used for biology storage (biobank) cell, microorganism, diseasePoison and other biological entity.There may be every chip-count thousand by the superchip device of the surface composition with high density microporeA, hundreds of thousands of or millions of a micropores.For example, can (or another biology be real by the microorganism from micropopulation sampleBody, such as different types of cell or virus) device is diluted and is applied to, so that hole includes about 1 microorganism/occupy hole.Then,Being incubated for chip replicates microorganism in hole, and gained group represents single type.Location index system based on nucleic acid is availableIn measuring in each hole, there are what types.The directory system may relate to for PCR primer to be loaded to each hole in advance.The PCR drawsObject contains the addressing bar code for identifying hole and provides in the targeted microorganisms genome of information or targeting target genetic sequenceThe primer sequence of specific genetic elements (such as 16S).After incubation, releasing microbe DNA, PCR primer expands non-target bacteria region of DNADomain.The amplicon in each hole on chip is collected, is then read by next-generation sequencing.
Using high density micro Process chip to carry out biological storage enables many types in each sample or variant as singlyOnly group's storage, without carrying out laborious separation scheme before storing or later.Use the location index based on DNASystem or custom analysis allow simpler general method to identify the heredity signature or feature of each micropore content, to provideInformation, such as information.In addition, chip apparatus provides a kind of method of effective torage cell isolate in very space.ExampleSuch as, the significant spatial that the chip of the single microslide size with 100,000 hole occupies is lower than corresponding conventional storeFormat.Chip format can also be by filing a chip suitably comprising many different samples from single subjectWith planning sample and/or management subject (for example, patient) information database.
Cell is stored to use the equipment, cell can be added and/or be placed in the micropore of the device.It can handleThen cell in micropore places it in suitable storage condition to mitigate the influence of storage.For example, can be for example sweet with reagentOil processing cell is to mitigate the influence of freezing.It is then possible to which chip is placed in suitable storage condition such as refrigerator.It can incite somebody to actionCell dehydration, freeze-drying, and/or freeze-drying, and chip can be placed in suitable condition to protect dry cell.It can be toAdditional structure is added further to promote it as the purposes of storage device in chip.For example, can be placed on chip film orAnother structural detail, to seal at least some holes before storing.
Cell can be added in chip, so that a part of micropore on chip is respectively occupied by each about cell.It incubatesChip is educated to allow cellular replication.Chip is replicated, comes in each hole of authentication chip to exist using the chip of original chip or duplicationCell or type or gene signature (by using above-mentioned location index system).By chip processing, and/or it is stored inUnder appropraite condition.Copy step and/or authentication step can after storing rather than carried out before storage.
One or more cells of each bacterial strain from one group of pre-existing isolated strains can be placed on chipIsolated micropore in.Can recorde the micro well locations that each bacterial strain or cell type are placed, and handle chip, and/or by itsStorage is under suitable conditions.
A kind of version of chip can be manufactured, wherein anti-corrosion chemicals are isolated under the wax barrier in each hole.PermitPerhaps isolate seals growth in the chip, discharges preservative by thermal induction before storage later to save.
Such equipment can be used for storing/mixed micropopulation the sample of biology storage, such as from soil, people's enteron aisle, seaThe micropopulation sample of water, oral cavity, skin etc..Chip can be used for storing other kinds of biological entities, such as fungi, Gu are carefullyBacterium, people's cell (including proliferative cell), zooblast and virus.
DNA location index system is suitable for all biological entities types, to generate the information about each hole content.It can be withEntire chip is screened using custom analysis (such as analysis based on antibody or substrate) to obtain expectation activity.For example, can sieveSelect the chip with the T cell group of storage to obtain specific immunocompetence.
It, can be from research participant collector fecal microorganism group's sample in an illustrative example.It can will come fromThe fecal microorganism biology of each individual is stored on chip, to retain the record of the individual micropopulation and later as targetMark microbe-derived micropopulation sample.In another example, during clinical or research or as treatment workflowA part (for example, using the cell therapy of ex vivo treatment cell), can be from individual sampling T cell or other immunocytesMictium.Also in another example, edaphone can be stored together with seed bank.
High-resolution is chosen
High-precision/accurate selected equipment or system can be designed with executed on above-mentioned micro Process chip from and/or toThe various selection functions of micropore.Target substrate or chip can be the microscope glass on a surface with injection moulding featurePiece format (about 25mm x 75mm x 1mm).Micropore can be arranged with mesh model, have about 4- in the perimeter of chip8mm without bore edges.Size and the interval in hole can be determined based on the ability of Chooser.Micropore can be is along each side sizeAbout 25 μm to about 200 μm of square, between bore edges between be divided into about 25 μm to about 100 μm.Micropore can be circle or sixSide shape.The depth in hole can be about 25 μm to about 100 μm.For example, edge of the slide of 75mm x 25mm with 7mm, 100 μmSquare hole, while while between be divided into 100 μm, have about 16,775 micropores.
High-precision/accurate selecting system can be designed executed on above-mentioned micro Process chip from and/or to corresponding toThe various selection functions of a part of film of micropore.Film is used to for the bacterium of growth being encapsulated into the thin slice of micropore before can be.WhenWhen removing from chip, film can retain the trace and bacteria samples of microwell array on the surface thereof after separating from chip.CauseThis, the film of removing can be used as the duplicate of the bacterium grown in the chips.
High-resolution Chooser can receive data input from user.Input may include that at least a pair of of chip micropore is satMark, so that Chooser is from least a pair of coordinate inputted and/or chooses the coordinate at least a pair of of input.It between cycles can be withCarry out sterilizing program.High-resolution Chooser can operate in anaerobic box.
High-resolution Chooser system can receive chip, and will for example be chosen using fiducial marker and/or reference boreDevice is aligned with chip.For example, Chooser can choose 96 of the cell grown in the micropore on chip extremely containing growth mediumHole or 384 orifice plates.Chooser may include single selection needle or multiple selection needles.
High-resolution Chooser system can receive film, and for example using the reference bore marker on film by Chooser and filmAlignment.For example, Chooser can choose cell to 96 holes containing growth medium or 384 orifice plates from film.Choosing needle can haveThere are different shape (such as mushroom-shaped) and/or surface (such as texture) to choose from film.System can also include one or moreThe mechanism (such as floating needle and/or vacuum) of a holding and/or flattening film.
High-resolution Chooser system can be via the transfer replication chip of chip to chip.Based on fiducial marker and/Or reference bore, Chooser can receive and be aligned the first chip.Based on fiducial marker and/or reference bore, Chooser can be withIt receives and is aligned the second chip.For example, the cell grown in the micropore on the first chip can be transferred to second by ChooserThe micropore of chip.
For being chosen at the target type of the multiple types for at least one biological entities cultivated in micromachining device automaticallyHigh throughput system may include interface for receiving micromachining device.Micromachining device defines high density microwell array.It is highEach micropore of density microwell array is equipped with to separate and cultivating at least one type of at least one biological entities, and including moreAt least one label of a unique label.Each label of the multiple unique label include nucleic acid molecules and with high density microporeThe relevant location specific nucleotide sequence of at least one micropore of array, the nucleic acid molecules include and at least one lifeThe target specificity nucleotide sequence of object entity annealing.System further includes that the high-resolution selection at least one protrusion is setIt is standby, it is used to choose at least one biological entities from least one micropore of high density microwell array.System further comprises usingIt is set in the input unit for receiving at least one target specificity nucleotide sequence instruction, and with input unit and high-resolution selectionAt least one processor of standby communication coupling.It is special that at least one described processor obtains at least one target from input unitThe instruction of specific nucleotide sequence, by least one described target specificity nucleotide sequence compared with multiple unique labels, baseAt least one micropore of the high density microwell array comprising target type is determined in comparison result, and is controlled high-resolution selection and setIt is standby to choose at least one biological entities from least one micropore determined of high density microwell array.
Disclose the target of multiple types for being chosen at at least one biological entities cultivated in micromachining device automaticallyMark the high throughput system of type.Micromachining device defines high density microwell array, each micropore of high density microwell array and moreAt least one unique primers of a unique primers are related.System includes the interface for receiving the film removed from micromachining device,Each micropore of film sealing high density microwell array at least one biological entities to be retained in high density microwell array,So that a part of at least one biological entities corresponding to high density microwell array is still adhered on film after removing film.SystemSystem further includes high-resolution selected equipment comprising: at least one protrusion, for from correspond to high density microwell array at leastAt least one film location of one micropore chooses at least one biological entities；Input unit is related to target type for receivingAt least one target specificity nucleotide sequence instruction；With communicated with input unit and high-resolution selected equipment couplingAt least one processor.At least one described processor obtains at least one target specificity nucleotides sequence from input unitThe instruction of column, by least one described target specificity nucleotide sequence compared with multiple unique labels, based on comparative result reallySurely at least one film location of at least one micropore corresponding to high density microwell array comprising target type, and control high scoreResolution selected equipment chooses a part of at least one biological entities from least one film location determined.
The embodiment above can be embodied in various ways.It is, for example, possible to use hardware, software, or its combinations to realize embodiment partyCase.When implemented in software, software code (either can individually calculated in any processor appropriate or processor setsThere is provided, be also distributed across in multiple computers in machine) on execute.
In addition, it should be understood that computer can embody in a variety of forms, such as frame type computer, desktop computer, penRemember this computer or tablet computer.It is not generally regarded as computer in addition, computer can be embedded in but there is proper treatmentIn the device of ability, including personal digital assistant (PDA), smart phone or any other suitable portable or fixed electronics are setIt is standby.
Also, computer can have one or more and output and input device.These devices can be used for presentation user circleFace.Can be used for providing the output device of user interface example include printer or for output vision present display screen andLoudspeaker or other flexible piezoelectric sound-generating devices of the sound presentation for output.It can be used for providing the reality of the input unit of user interfaceExample includes keyboard, indicator device such as mouse, touch tablet and digitizer tablet.As another example, computer can pass through soundSound identification or other form of sound receive input information.
This computer can in any appropriate form by one or more network interconnections, including such as enterprise network,The local area network or wide area network of intelligent network (EST) or internet etc.This network can be based on any technology appropriate, and can be withIt is operated according to any scheme appropriate, and may include wireless network, cable network or fiber optic network.
The various methods or process listed herein can be encoded to software, can use various operating systems or platformOne of any one or more processors on execute.In addition, this software can use any various suitable programming languagesAnd/or programming or wscript.exe are write, and the executable machine language code executed on a framework or virtual machine can also be compiled asOr intermediate code.
Further example
In an example, film is used for " separated " micro Process chip as described herein, to generate from binary mixtureIsolate.Previous evening starts fresh culture.It is being added to before on chip, bacterium is counted (Analyzer 10, Miltenyi Biotec), dilute and mix, to generate the calcium acetate of equal portions notThe mother liquor of lever bacterium (AC) and saccharomyces cerevisiae (SC).Selecting the two components is because they are easy in typical inverted microscopeIn differentiation.
AC/SC mixture is diluted to the cell for targeting and separating in each micropore (for each containing microorganismMicropore).It then, will be diluted by the way that mixture (or solution, suspension) to be moved on the microwell array on chip surfaceAC/SC mixture is added on the micropore to chip.Since this is a random adition process, some micropores are added one thinBorn of the same parents, some micropores are empty, and more than one cell is added in each micropore of some micropores.After micropore is added, back is usedPolycarbonate membrane seal array with PCR band.Apply strong pressure to ensure that micropore is separated from each other.Then by chip at 30 DEG CIt is incubated overnight.At second day, check chip to provide the evidence of the excellent sealing of growth and micropore by inverted microscope.It usesCommon split approach in Fiji (open source image procossing packet), together by the Image Mosaic of chip.Figure 24 A shows composite diagramPicture is stitched together by a series of images obtained from the inverted microscope of chip (appropriate to have storage cavern and film).Figure 24 B isThe close-up image obtained from inverted microscope, which show the micropores of the micropore of some growths and some skies.Figure 25, which is shown, to be directed toThe MIcrosope image for each micropore that pure culture (SC, AC and their mixture) is observed.
After growing a period of time on chip, culture medium storage cavern is removed and carefully by film stripping with separating chips.It will strippingFrom film be installed in order to handle on back lining materials, and store until getting out picking.
Chip is observed for the micropore containing pure culture.Identify 8 holes, each type 4, and determine them on filmRespective positions (X and Y coordinates).Each micropore leaves the apparent marking on film, allows to micropore (such as the cross from the upper right cornerIt is to three and two downward) it counts and positioning covers and seals the part on the film of the particular bore.
Using the small needle on 3 axis controllers being mounted on inverted microscope objective table, film is touched in selected location first,Then needle is immersed in culture base tube to generate pure culture.Between picking, needle is disinfected in alcohol.
Eight tubules are incubated for 48 hours at 30 DEG C, are then checked.Pollution is not observed.Three in four AC pickingsOne in a and four SC pickings is successful.Figure 26 shows the pure culture (in Figure 26 A) of SC and the pure culture (figure of ACIn 26B) image.
In an example, micro Process chip as described herein is used for high flux screening.Two e. coli strains are (a kind ofCan be lactic acid (lac+) by lactose metabolism, and one kind cannot (lac-)) it mixing, dilutes and spreads on chip.Lac+ bacteriumExist with 1% abundance, and gross sample is diluted downwards to provide and each occupy about 1, hole cell.By bacterium in poly- carbonic acidIt is sealed under ester film, silicone gasket is then fixed on array.Eosin methylene blue (EMB) agar of temperature is poured into washer and madeIt is hardened above array.EMB agar has selectivity to certain form of gramnegative bacterium, and provides for lactose fermentationColorimetric instruction.Lactose fermentation can be reduced into local ph at the bacterium of lactic acid, increase the absorption of dyestuff, these bacterium colonies is made to become deepPurple or black.Non-zymocyte will not reduce pH value, in some instances it may even be possible to will increase pH value.Those bacterium colonies keep transparent.Keep chip rawIt is 48 hours long.Then it shoots under the microscope.By 14 image combinations to show a large amount of 50 micron openings on array.
Image shown in Figure 22 A shows that there are lac+ bacteriums wherein 9 dimmed comprising 1800 micropores.In Figure 22 BThe image shown is the amplifier section of image shown in Figure 22 A, shows the appearance of dark micropore.
In another experiment, " counting " of chip is obtained.Then EMB agar on film uses needle for measuring dark holesAgar layer and film are pierced through, and material is transferred to tubule from micropore, and start new bacterium colony.
In some examples below, the bacterium gDNA of purifying is used to carry out PCR on the chip of the disclosure as sample.In this process, by the PCR pre-composition (mastermix) containing 16S rRNA V4 primer (for the chip of oligonucleotides printingPrimer, is not added into pre-composition by (oligo printed chips)) and template gDNA be moved to chip surface on.It willStorage cavern is placed on the micropore on chip, and adds mineral oil to avoid the evaporation of PCR buffer.Used thermocycling program is96 DEG C 10 minutes, 39 circulation 60 DEG C 2 minutes, 98 DEG C 40 seconds, 60 DEG C 2 minutes, then keep 10 DEG C.After PCR, core is taken outPiece goes oil removing, amplicon is washed off from chip, and extract 16S rRNA V4 amplicon with Qiagen Qiaquick kit.Then by pipe PCR, with NGS primer amplification these amplicons indexed based on sample, to prepare the library NGS.Pipe PCR programFor 95 DEG C 2 minutes, 35 circulation 95 DEG C 35 seconds, 50 DEG C 45 seconds, 72 DEG C 45 seconds.Then 72 DEG C 2 minutes, and kept for 4 DEG C.GelElectrophoresis is used to determine the presence of amplicon.
In some following instances, bacterial cell is used to carry out PCR on the chip of the disclosure as sample.In the processIn, by being moved to bacterial suspension on the chip surface where micropore, bacterium is added to chip.Film is applied to coreOn piece is incubated for chip with the bacterium in sealing hole.At 30-37 DEG C after incubation 1-5 days, (incubation time and temperature depend on usedMicrobe species), the bacterium in micropore is centrifuged, goes membrane removal, and by gently blotting chip with absorption paper to absorb trainingSupport base.By containing 16S rRNA V4 primer PCR pre-composition (for oligonucleotides printing chip, not by primer be added intoPre-composition) it is moved on the surface of chip.Storage cavern is placed on the micropore on chip, and it is slow to avoid PCR to add mineral oilFliud flushing evaporation.Used thermocycling program be 96 DEG C 10 minutes, 39 circulation 60 DEG C 2 minutes, 98 DEG C 40 seconds, then 60 DEG C2 minutes and 10 DEG C of holding.After PCR, chip is taken out, amplicon is washed off from chip, and with Qiagen Qiaquick kitExtract 16S rRNA V4 amplicon.Then by pipe PCR, with NGS primer amplification these amplicons indexed based on sample,To prepare the library NGS.Pipe PCR program be 95 DEG C 2 minutes, 35 circulation 95 DEG C 35 seconds, 50 DEG C 45 seconds, 72 DEG C 45 seconds.Then72 DEG C 2 minutes, and keep 4 DEG C.Gel electrophoresis is used to determine the presence of amplicon.
Escherichia coli gDNA is used to be tested as target.PCR is added in Escherichia coli gDNA, primer and archaeal dna polymeraseIn buffer, it is used for two different micropore sizes (100 μm and 400 μm).Design primer is to expand the region 16S of DNA of bacteria.Negative control is identical process, but does not have archaeal dna polymerase in reaction buffer.By chip PCR, QiagenAfter the second wheel PCR in Qiaquick kits and pipe, the results showed that exist corresponding to 16S V4 amplicon (T1-1, T1-2) band, but not negative control (T2-1, T2-2).This confirms that 16S rRNA V4 segment expands on chip really.100μThe hole of m shows as having preferably amplification (strong band).On the data validation chip in the region 16S from Escherichia coli gDNAAmplification.
In a further test, genomic DNA (20 kinds of bacteriums, BEI from microorganism simulation group B are usedResources HM-782D, table 8) as the target sample for PCR on chip.Chip PCR and the library NGS indexed with sample(Figure 27 shows the gel electrophoresis in the library NGS, and swimming lane 1-2 shows the amplicon of 16S rDNA V4 after amplification.Swimming lane 1, from simulationThe amplicon of group gDNA；Swimming lane 2, the amplicon from simulation group gDNA；Swimming lane 3, negative control；Swimming lane 4, DNA ladder shapeBand), NGS is run on Illumina MiSeq.
8. microorganism of table simulates group B
NGS data pass through mass filter with about 2550596 total indicator reading and 93%.From boundary to kind, total indicator reading classificationFor 57% (see Figure 28) of the horizontal 72-73% of taxology and kind.NGS data are shown in addition to propionibacterium acnes, it is possible to identify the mould95% category that quasi-group is fallen.By 16S sequence, 17 kinds (85%) in total in 20 kinds are identified with the level of kind.Table 9 shows logicalHorizontal 17 kinds identified from gDNA simulation group that 16S NGS analysis is crossed to plant.Other two kind (Liszt's formula bacterium and newborn barBacterium) it is identified with the level of category.Do not identify kind and call fusobacterium may be related to Clostridium beijerinckii.
The chip PCR NGS data analysis of 9. pairs of the table categories for simulating group and kind classification
| Boundary | Door | Guiding principle | Mesh | Section | Belong to | Kind | It counts | Count % |
| Bacterium circle | Thermus door | Abnormal cocci guiding principle | Abnormal cocci mesh | Abnormal cocci section | Deinococcus | Anti-radiation kind | 186727 | 14.168 |
| Bacterium circle | Firmacutes | Bacillus guiding principle | Bacillus mesh | Listeria Cordycepps | Listeria | Harmless kind | 80765 | 6.128 |
| Bacterium circle | Proteobacteria | ε-deformation Gammaproteobacteria | It is bent Zoopagales | Screw rod Cordycepps | Screw rod Pseudomonas | Pylorus kind | 51573 | 3.913 |
| Bacterium circle | Firmacutes | Bacillus guiding principle | Lactobacillus mesh | Streptococcaceae | Streptococcus | Deformation kind | 42768 | 3.245 |
| Bacterium circle | Proteobacteria | β-deformation Gammaproteobacteria | Neisser Zoopagales | Neisseriaceae | Neisseria | Meningitis kind | 34539 | 2.621 |
| Bacterium circle | Firmicutes | Bacillus guiding principle | Lactobacillus mesh | Streptococcaceae | Streptococcus | Agalasisa kind | 30704 | 2.33 |
| Bacterium circle | Firmicutes | Clostridium guiding principle | Clostridium mesh | Clostridiaceae | Fusobacterium | | 23726 | 1.8 |
| Bacterium circle | Firmicutes | Bacillus guiding principle | Lactobacillus mesh | Lactobacillaceae | Lactobacillus | Taiwan kind | 23293 | 1.767 |
| Bacterium circle | Bacteroidetes | Bacteroid guiding principle | Bacteroid mesh | Bacteroides | Bacteroides | Common species | 23258 | 1.765 |
| Bacterium circle | Proteobacteria | γ-deformation Gammaproteobacteria | Pseudomonadales | Moraxella section | Acinetobacter | Bao Man kind | 16792 | 1.274 |
| Bacterium circle | Firmicutes | Bacillus guiding principle | Bacillus head | Staphylococcaceae | Staphylococcus | Golden yellow kind | 12022 | 0.912 |
| Bacterium circle | Proteobacteria | α-deformation Gammaproteobacteria | Red bacterium mesh | Red bacterium section | Red bacterium category | Class ball kind | 9574 | 0.726 |
| Bacterium circle | Proteobacteria | γ-deformation Gammaproteobacteria | Pseudomonadales | Pseudomonadaceae | Pseudomonas | Verdigris kind | 7155 | 0.543 |
| Bacterium circle | Proteobacteria | γ-deformation Gammaproteobacteria | Pseudomonadales | Enterobacteriaceae | Escherichia | Large intestine kind | 6849 | 0.52 |
| Bacterium circle | Actinomyces door | Actinomycetes | Actinomycetal | Actinomy cetaceae | Actinomyces | Molten tooth kind | 3558 | 0.27 |
| Bacterium circle | Firmicutes | Bacillus guiding principle | Bacillus head | Staphylococcaceae | Staphylococcus | Epidermis kind | 1733 | 0.131 |
| Bacterium circle | Firmicutes | Bacillus guiding principle | Lactobacillus mesh | Streptococcaceae | Streptococcus | Pneumonia kind | 656 | 0.05 |
| Bacterium circle | Firmicutes | Bacillus guiding principle | Lactobacillus mesh | Lactobacillaceae | Lactobacillus | Jia Shi kind | 298 | 0.023 |
| Bacterium circle | Firmicutes | Bacillus guiding principle | Bacillus head | Series bacillus section | Bacillus genus | Prosopidis kind | 233 | 0.018 |
| Bacterium circle | Firmicutes | Clostridium guiding principle | Clostridium mesh | Clostridiaceae | Fusobacterium | Bai Shi kind | 179 | 0.014 |
In another test, chip PCR is carried out with simulation group's bacterial cell to NGS.It grows in 7 bacterium kinds conductsSimulate group in portion comprising: pseudomonas aeruginosa, salmonella typhimurium, Staphylococcus warneri, serratia marcesens, waxy budSpore bacillus, bacillus subtilis and Escherichia coli.To chip PCR, according to general process same as described above.Simulation group is mixedIt closes object to be added to chip, be sealed with film, is incubated overnight, by chip with 3000rpm centrifugation 3 minutes.Membrane removal is gone, it is then net with wipingPaper absorbs culture medium.Chip is filled with PCR buffer and 16S rRNA PCR primer, and thermal cycle is carried out according to the above method.NGS DNA library is prepared using process as described above, and is sequenced with Illumina Miseq.Figure 29 is shown from amplification sampleWeak amplicon band.6 categories of gained NGS data analyzed and identified from internal simulation community.It is identified and is come from the level of kind3 kinds for simulating group.
In another example, including bar code (" location tags ") system for being printed onto the micropore of chip.i5Index and i7 index be designed to be connected to 16S rRNA V4 probe forward primer and reverse primer (referring to Figure 30, whereinIt indexes i5 and i7 and is used for chip hole site, and index sample index of the i7 ' for Multi-example measurement.Each index is 8-12 longBetween base-pair, and we devise different combinations, to identify each hole in 500 microwell chips 2.Using commercially availableAccurate printing instrument print two oligonucleotides (each oligonucleotides contains an index sequence and 16S primer sequence) to everyIn a micropore (100 μm of 100 μ m of micropore size, 100 μ m).Each micropore contains different index combinations, therefore directory systemIt provides for identifying the PCR product derived from each micropore.After saving PCR primer, using in printing equipment and this specificationWax layer is added into each micropore by the method described elsewhere.In addition, sample directory system is designed to allow in NGSEach sample is marked in the preparation process of library.Each sample index has 8-12 base-pair.It is added during second step PCREnter product index.Therefore, single NGS operation can be from the DNA formation sequence data tested derived from multi-chip, and are connected to generationThe chip of DNA.This can save the time and reduce the NGS cost of each sample.
As described above, the oligonucleotides of index and wax printing are brushed in the micropore of chip.By PCR pre-composition (primer free) andSimulation group gDNA is added into chip for running chip PCR.It is each chip amplification in the second wheel PCR after chip PCRSubsample is added into product index and NGS sequence measuring joints.Run four chips and using four different sample indexes.It usesMiseq (Illumina, San Diego) carries out NGS to all four samples in primary operation.Using computer program to thisA little indexes are decoded, and extract the NGS data from this four samples.Sample data shows that NGS success has read across chipMicropore inherently indexes.37% hole, which has, is greater than 300 sequence reads (> x300 sequence, 925 holes), and 25% hole has100-299 sequential calling, 28% hole have 1-99 sequential calling, and only 10% hole has 0 sequential calling (ginsengSee Figure 31).These results prove that printer chip has double index oligonucleotides workflows.
In one aspect, a kind of method of at least one of screening sample target biological entities is provided.The method usesMicromachining device as described herein has the top surface for defining microwell array.At least one micropore of microwell array is added:(a) at least one cell from sample；(b) indicator substance；With (c) a certain amount of nutrients.Cell, indicator substance and battalionFeeding object can sequentially add in any order, or any combination of them can be sequentially or simultaneously added.Film is applied toMicromachining device is at least one cell to be retained at least one micropore.It is permeable that film can be only gas (such as oxygen)Film or fluid permeable or impermeable film.The component of nutrients can penetrate into inside micropore by film outerPortion, or nutrients storage cavern can be provided outside film and outside micropore, and nutrients can penetrate into micropore by film.Micromachining device is incubated in predefined conditions (for example, in desired temperature/atmosphere and under being suitable for the other conditions of cell)For a period of time, to grow multiple cells from least one cell at least one micropore.At least one micropore can be evaluatedOptical characteristics, such as color, optical density, fluorescence, phosphorescence or luminous.Optical characteristics can be evaluated as the collection of each independent microporeBody or average characteristics, or can be evaluated in more detail in each hole for example as optical characteristics pattern, such as optical morphology.The evaluation can be carried out when being incubated for completion and/or at multiple time points, such as after micropore content is added but be incubated forBefore and be incubated for started after but not yet complete etc..It is evaluated based on the optical characteristics assessed or multiple optical characteristicsBetween compare, can determine at least one micropore presence or absence of at least one target biological entities.
As used herein, " indicator substance " refers to before incubation, is incubated for after completion, and/or in incubation processIn but before being incubated for and completing, make micropore locating for it that there is the chemical substance of certain different optical characteristics.Such opticsCharacteristic may due in object micropore indicator substance and cell react or interaction (such as moves in cell, thinBorn of the same parents' intake etc.), with cellular component react interaction (such as be incorporated into cell membrane, the DNA for being incorporated into cell etc.) or byCell generate substance and change.The variation of optical characteristics may be due to the amount of indicator substance variation (such as its part orAll due to being converted into different compounds from the reaction of cellular component or cellular products) or indicator substance structure weightGroup or indicator substance are incorporated into another component.
In some embodiments, indicator substance can be inorganic phosphate compounds, such as calcium phosphate, not dissolve inWater.When being stored in micropore and by micro- sem observation, it can be observed that calcium phosphate granules.The compound can be used for screeningCertain acid be can produce so that the phosphate-solubilizing bacteria that compound dissolves.As a result, phosphate can be reduced or be disappeared.It can also makeWith other inorganic insoluble phosphates, such as aluminum phosphate.It can be for example by capturing the digital picture of micropore under the microscope simultaneouslyThe comparison of micropore is carried out by the variation of the color of computer software analysis image, optical density, pattern or other features.
In an example of the embodiment, by printing designated volume and concentration calcium source (such as calcium chloride) andCalcium phosphate is stored in the micropore of micromachining device by phosphate source (such as sodium phosphate) using accurate printing equipment.With the partyFormula, starting material are the liquid for being easy to distribute, and are then reacted in the bottom of each micropore to form solid phosphoric acid calcium.The result is thatThere is the micromachining device of the calcium phosphate of controlled quatity in the bottom in each hole.By bacterial strain (one is dissolving P capacity be the positive, oneKind is that dissolving P capacity is negative) dilution, so that micropore contains single bacterium and some nutrients.Film is applied using pressure and heatIt is added on the top surface of micromachining device, seals the content of micropore.Micromachining device is incubated overnight to grow more many cells.Micromachining device is observed in various time points under an optical microscope.It is to be easy mark containing the micropore for capableing of phosphorus decomposing bacterial strain,Because of the disappearance when making it dissolve of the calcium phosphate of hole bottom.As shown in Figure 32 A-32D, each hole is shown under high-amplification-factor:Because without cell presence, phosphate is constant (in Figure 32 A)；Phosphorus decomposing cell has grown and has dissolved phosphate (Figure 32 BIn)；Non- phosphorus decomposing cell has been grown but phosphate does not change (Figure 32 C, 32D).
The position for each micropore that calcium phosphate disappears can be supplied to picking device, the picking device can be used for byAt least part content of micropore is transferred to target position, such as 96 orifice plates for being further incubated for/growing.It is raw in 96 orifice platesAfter length, the amount of material is sufficient to various further tests and identification procedure.
In some embodiments, indicator substance can be pH sensitive dye.For example, pH sensitive dye can be selectedChange color within the scope of pH in visible-range.PH sensitive dye can also fluoresce within the scope of the first pH, andIt does not fluoresce within the scope of two difference pH.When pH changes, its fluorescent characteristics also can change, such as absorb the wave of incident lightLength, transfer efficiency, again launch wavelength etc..PH sensitive dye can be saved in micropore respectively, or be included in growth microorganismCulture medium or nutrients in.If acid or alkali are generated in the growth and/or breeding of cell in micropore, the dyestuffColor may change.
In the example of the embodiment, pH sensitive dye such as chlorophenol red, bromocresol purple and bromthymol blue are used asIndicator substance.In an example, using bromocresol purple, from pH7 (or more) purple become the Huang of pH5 (and following)Color.It includes in the culture medium or nutrients of growth microorganism.Bacterial strain is added to the micromachining device with nutrientsMicropore in, sealed with film, and make its growth overnight.Micromachining device is observed in various time points under an optical microscope.FromColor change (by naked eyes or utilizing the image analysis of computer software) in the micropore observed, identifies containing generation shadowRing those of the bacterial strain of chemicals of pH micropore.
In some embodiments, the chemiluminescence dye, such as luminol of pH sensitivity are used as indicator substance.?In further embodiment, luminescence generated by light (the fluorescence or phosphorescence) dyestuff or material of pH sensitivity are used as indicator substance(or a part of indicator substance).For example, pH sensitive fluorescence dye can fluoresce within the scope of the first pH and differentIt does not fluoresce within the scope of two pH.In another example fluorescent dye can have different fluorescent characteristics at different pH.In phosphor materialIn the case where, phosphor can be coated with there are many different pH sensitive dyes.Due to the phosphor from excitation to coated dyeThe energy transfer of material, absorbing most dyestuffs under given local pH will emit brightest.
In some embodiments, indicator substance may include oxidation-reduction indicator, and optical characteristics can depend onSpecial electrodes current potential.For example, resazurin (7- hydroxyl -3H- phenoxazine -3- ketone 10- the oxidation as blue dyes and hypofluorescenceObject) the fluorescence resorufin of pink and height red can be reduced into.Resazurin is used as indicator substance, micro- with determinationWhether living bacterium is contained in hole.
Micromachining device may include a series of substrate with functional layers.The functional layer of the series includes defining firstFirst functional layer of array experiment unit (such as hole) and subsequent experimental considerations unit is defined in each experimental considerations unit of functional layer beforeAt least one subsequent functional layer of array (such as micropore).Each experimental considerations unit can be set to receive and grow at least one carefullyBorn of the same parents are screened at least once, and/or test at least one nutrients.
In one embodiment, the equipment for screening the different condition for being directed to cellular matrix includes substrate.Substrate packetInclude first surface.The hole of first array of delimited.Each hole includes inner surface.Each inner surface defines the micro- of second arrayHole.Each micropore is set as receiving and growing at least one cell.
In one embodiment, the equipment for screening the different condition for being directed to cellular matrix includes having a series of functionThe substrate of ergosphere.The functional layer of the series includes: the first functional layer, defines the experimental considerations unit of the first array, and at least oneA subsequent functional layer defines the experimental considerations unit of the subsequent array of each experimental considerations unit of aforementioned functional layer.Each experimental considerations unitIt is set as receiving and growing at least one cell.
In one embodiment, it discloses for point cellifugal method in the substrate for including first surface.FirstThe experimental considerations unit of the first array of delimited.Each experimental considerations unit is set as receiving at least one cell.At least each experimental considerations unitA part be set as with first surface feature comprising attract cell and/or increase cell and occupy the tendency of experimental considerations unit.Or or in addition, a part of at least first surface is set as with second surface feature comprising repel cell and/or reductionThe tendency of cell adherence on the first surface.Method further includes to composition of the first surface application including cell, so that at leastOne cell occupies at least one experimental considerations unit.
In one embodiment, the method for sampling the cell mass in substrate includes by based first surfaceCell mass is sampled at least one micropore using selecting device.Described device includes at least one in face of the prominent of first surfaceIt rises.The diameter of at least one protrusion is less than the opening diameter of each micropore.At least one protrusion insertion accommodates cell massAt least one micropore in so that a part of cell mass attachment at least one micropore and/or being adhered at least one protrusion.Method further includes taking out the cell mass sample at least one micropore by removing device from the first surface of substrate, so that extremelyA part of cell mass in a few micropore still adheres to and/or is adhered at least one protrusion.
In one embodiment, the method for sampling the cell mass in substrate includes by based first surfaceCell mass is sampled at least one experimental considerations unit using selecting device.Described device includes at least one in face of first surfaceProtrusion.The diameter of at least one protrusion is less than the opening diameter of each micropore.At least one protrusion insertion accommodates cellGroup at least one experimental considerations unit in so that at least one experimental considerations unit a part of cell mass attachment and/or be adhered toA few protrusion.Method further includes thin at least one experimental considerations unit to take out by removing device from the first surface of substrateBorn of the same parents' group's sample, so that a part of cell mass at least one test unit still adheres to and/or be adhered at least one protrusion.
In one embodiment, for sampling in the base including first surface and the second surface opposite with first surfaceThe method of cell mass in bottom includes based second surface application selecting device, and the first surface defines the first arrayMicropore.Described device includes the protrusion that at least one faces second surface.The diameter of at least one protrusion is approximately equal to or smallIn the opening diameter of each micropore.At least one described protrusion is compressed against on the second surface and accommodates at least the one of cell massThe corresponding position of a micropore, and/or insertion accommodate at least one micropore of cell mass, so that one at least one microporePart cell mass is moved on the inner surface in hole and/or on the first surface of substrate.Method further includes by collecting movable partCell mass sample the cell mass at least one micropore.
In one embodiment, for sampling in the base including first surface and the second surface opposite with first surfaceThe method of cell mass in bottom includes based second surface application selecting device, and the first surface defines the first arrayExperimental considerations unit.Described device includes the protrusion that at least one faces second surface.The diameter of at least one protrusion is approximately equal toOr the opening diameter less than at least one experimental considerations unit.At least one described protrusion is compressed against on the second surface and receivingThe corresponding position of at least one experimental considerations unit of cell mass, and/or insertion accommodate at least one experimental considerations unit of cell mass,So that a part of cell mass at least one experimental considerations unit is moved on the first surface of substrate.Method further includes collecting movementPartial cell mass.
In one embodiment, the method for sampling the cell mass in the substrate for including first surface includes Xiang WeijiaThe first surface application selecting device of work substrate, the first surface define the hole of the first array, and the inner surface in each hole definesThe micropore of two arrays, each micropore have opening diameter.Described device includes the protrusion that at least one faces first surface.It is described extremelyThe diameter of a few protrusion is less than the diameter of each micropore.At least one protrusion insertion accommodates at least one micropore of cell massIn, so that the long-pending inner surface for being moved to hole of a part of population of cells at least one micropore and the first surface of substrate are at leastOn one.Method further includes the cell mass sampled at least one micropore by the cell mass of collected volume movable part.
In one embodiment, the method for sampling the cell mass in the substrate for including first surface includes Xiang WeijiaThe first surface application selecting device of work substrate, the first surface define the experimental considerations unit of the first array.Described device includesAt least one faces the protrusion of first surface.The diameter of at least one protrusion is less than the diameter of at least one experimental considerations unit.At least one protrusion insertion accommodates at least one experimental considerations unit of cell mass, so that one at least one experimental considerations unitPart population of cells product is moved on the first surface of substrate.Method further include by the cell mass of collected volume movable part comeSample the cell mass at least one experimental considerations unit.
In one embodiment, for sample include first surface substrate in cell mass method include pass through toThe first surface application selecting device of substrate samples the cell mass at least one micropore, and the first surface defines first gustThe hole of column, each hole include inner surface, and each inner surface defines the micropore of second array, and each micropore has opening diameter.Described deviceAt least one needle and/or nanopipette including facing first surface.The overall diameter of at least one described needle and/or nanopipetteLess than the opening diameter of each micropore, and its interior diameter can accommodate target cell diameter.At least one needle and/or nanopipette insertionIn at least one micropore for accommodating cell mass.Method further includes by a part of cell mass by using pressure from least one microporeIntroducing device takes out the cell mass sample at least one described micropore.
In one embodiment, for sample include first surface substrate in cell mass method include pass through toThe first surface application selecting device of substrate samples the cell mass at least one micropore, and the first surface defines first gustThe experimental considerations unit of column.Described device includes at least one needle and/or nanopipette in face of first surface.At least one described needleAnd/or the overall diameter of nanopipette is less than the opening diameter of each micropore, and its interior diameter can accommodate target cell diameter.At least oneA needle and/or nanopipette insertion accommodate at least one experimental considerations unit of cell mass.Method further includes will by using pressureA part of cell mass takes out the cell mass sample at least one described experimental considerations unit from least one experimental considerations unit introducing deviceProduct.
In one embodiment, for sample include first surface substrate in cell mass method include pass through toAccommodate the acoustic energy of at least one micropore application focusing of cell mass in a liquid to sample the liquid at least one microporeIn cell mass, the first surface defines the hole of the first array, and each hole includes the inner surface for defining the micropore of second array.It is poly-Burnt acoustic wave energy by effectively from least one micropore spray drop in a manner of application.Drop includes thin at least one microporeBorn of the same parents' group's sample.
In one embodiment, for sample include first surface substrate in cell mass method include pass through toAccommodate the acoustic energy of at least one experimental considerations unit application focusing of cell mass in a liquid to sample at least one experimental considerations unitIn liquid in cell mass, the first surface defines the experimental considerations unit of the first array, the acoustic wave energy of focusing with effectively fromThe mode application of at least one experimental considerations unit injection drop.Drop includes the cell mass sample at least one experimental considerations unit.
In one embodiment, substrate includes first surface and second surface.First surface defines the hole of the first array.Each hole has the inner surface for the micropore for defining second array.Substrate includes at least first and second, and described first includesAt least part first surface, described second includes at least part second surface.Described first and second along extremelyFew a part plane parallel with the first surface and second surface is detachably connected.The plane divides the micro- of second arrayHole.First array and/or second array can be substantially planar.For sampling the side of the cell mass at least one microporeMethod includes disassembly first and second, so that the cell mass of the first part at least one micropore is still adhered to firstPiece, and the cell mass of the second part at least one micropore is still adhered to second.
In one embodiment, substrate includes first surface and second surface.First surface defines the reality of the first arrayVerification certificate member.Substrate include at least first and second, described first include at least part first surface, described secondIncluding at least part second surface.Described first and second along at least part and the first surface and the second tableThe parallel plane in face is detachably connected.The plane divides the experimental considerations unit of the first array.First array can be substantially flatFace.Experimental considerations unit can be hole.Method for sampling the cell mass at least one experimental considerations unit includes disassembly firstWith second so that the cell mass of the first part at least one experimental considerations unit is still adhered to first, and at least oneThe cell mass of second part in experimental considerations unit is still adhered to second.
In one embodiment, substrate includes defining the first surface in the hole of the first array, and each hole includes inner surface, respectivelyInner surface defines the micropore of second array.Detachable film is applied at least part of at least one inner surface, so that at least oneA part of cell mass in a micropore is attached to the detachable film.Method for sampling the cell mass at least one microporeIncluding removing the detachable film, so that a part of cell mass at least one micropore is still adhered to the detachable film.
In one embodiment, substrate includes defining the first surface of the experimental considerations unit of the first array.To at least onePoint first surface applies detachable film so that a part of cell mass at least one experimental considerations unit be attached to it is described detachableFilm.Method for sampling the cell mass at least one experimental considerations unit includes the removing detachable film, so that at least oneA part of cell mass in experimental considerations unit is still adhered to the detachable film.
In one embodiment, substrate includes first surface and second surface.First surface defines the hole of the first array.Each hole has inner surface, and each inner surface defines the microchannel of second array.Each microchannel has the first opening in first surfaceWith the second opening in second surface.The first detachable film is applied at least part of at least one inner surface, so that at leastAt least some of one microchannel cell mass is attached to the first detachable film.Second is applied at least part of second surfaceDetachable film, so that at least some of at least one microchannel cell mass is attached to the second detachable film.For sampling at leastThe method of cell mass in one microchannel includes the first detachable film of removing, so that at least some of at least one microchannelCell mass is still adhered to the first detachable film, and/or the second detachable film of removing, so that at least one microchannel at leastSome cell masses are still adhered to the second detachable film.
In one embodiment, substrate includes first surface and second surface.First surface defines the reality of the first arrayVerification certificate member.Each experimental considerations unit has the first opening in first surface and the second opening in second surface.To at least partFirst surface applies the first detachable film, so as to be attached to first removable at least some of at least one experimental considerations unit cell massUnload film.The second detachable film is applied at least part of second surface, so that at least some of at least one experimental considerations unitCell mass is attached to the second detachable film.Method for sampling the cell mass at least one experimental considerations unit includes removing firstDetachable film, so that at least some of at least one experimental considerations unit cell mass is still adhered to the first detachable film, and/or strippingFrom the second detachable film, so that at least some of at least one experimental considerations unit cell mass is still adhered to the second detachable film.
In one embodiment, the method for cultivating the cell being originated from the sample of environment includes: based theSample is applied on one surface, so that at least one cell occupies at least one micropore, hole or experimental considerations unit；To at least part firstSurface apply semi-permeable film, allow nutrients to diffuse at least one micropore, hole or experimental considerations unit, and prevent and/orMitigate at least one and occupy cell and is fled from from least one micropore, hole or experimental considerations unit；And at least one nutrientsAt least one occupies cell described in being incubated at least one micropore, hole or experimental considerations unit.
In one embodiment, for cultivating and adapting to the method packet of the cell in the sample from environment in the substrateIt includes: based first surface application sample, so that at least one cell occupies at least one micropore, hole or experimental considerations unit；ToAt least part first surface application semi-permeable film allows nutrients to diffuse at least one micropore, hole or experiment singleMember, and prevent from and/or mitigate at least one and occupy cell to flee from from least one micropore, hole or experimental considerations unit；Have at leastBe incubated in a kind of at least one micropore of nutrients, hole or experimental considerations unit it is described at least one occupy cell；Use progressive portionExchange is divided to be gradually transitions other at least one nutritional preparations from least one nutrients whithin a period of time；And it detectsThe growth of cell is occupied at least one of at least one micropore, hole or experimental considerations unit.
In one embodiment, combined analysis of molecules data element can be distributed and/or it is related return it is original eachA position.In one embodiment, microarray includes multiple positions for applying sample.Each position on microarray can be withIt is set as receiving a part of sample.Each position is marked with unique label, the unique label for identify a part of sample fromMicroarray remove after, the sample segment from position.
In one embodiment, it is moved in a part of sample containing at least one nucleic acid molecules from microarray for identifyingAfter removing, the method for which position of the sample segment on microarray includes step (a) in multiple positions of microarrayThe sample of one or more parts is applied in one or more.Each position is marked with the unique label comprising nucleic acid molecules.It is describedNucleic acid molecules include: (i) location specific nucleotide sequence；(ii) first target specificity nucleotide sequence.This method is alsoThe nucleic acid molecules found and the label of mark position at least part sample is allowed to anneal including step (b).This method is into oneStep includes that step (c) carries out primer extend, reverse transcription, single-stranded connection or double-strand connection to the nucleic acid molecules group of annealing, thus willLocation specific nucleotide sequence is integrated into each core generated by primer extend, reverse transcription, single-stranded connection or double-strand connectionAcid molecule.This method further comprises that step (d) merges the nucleic acid molecules group generated in step (c)；(e) combined nucleic acid is sequencedMolecular group, to obtain the sequence of one or more location specific nucleotide sequences；It (f) will be from combined nucleic acid molecules groupThe sequence of at least one the location specific nucleotide sequence obtained with the label mark comprising location specific nucleotide sequencePosition on the microarray of note is associated, so that a part of sample of the identification comprising at least one nucleic acid molecules is from microarrayWhich position.Sample may include at least one cell, and cell can replicate after step (a) and before step (b).?Before step (b), a part of a part of sample, and a part of a part of sample can be removed from least one positionIt can store in independent container relevant to the initial position of a part of sample described on microarray.
It in one embodiment, include including for the method for the microarray for applying multiple positions of sample for manufacturingStep (a) synthesizes multiple labels, and wherein at least one position is marked with unique label.Each label includes nucleic acid molecules, describedNucleic acid molecules include: (i) location specific nucleotide sequence；(ii) first target specificity nucleotide sequence.This method intoOne step includes at least one position for multiple positions that label is placed in microarray by step (b).Unique label may include coreAcid molecule, the nucleic acid molecules include: (i) location specific nucleotide sequence；(ii) first target specificity nucleotides sequenceColumn.Label may further include amplimer binding site and/or aptamer nucleotide sequence.
Cyclic olefin polymer can be used via injection moulding to manufacture micro Process chip.Substrate or chip can have baseFlat surface in sheet, size are about 1 inch and multiply about 3 inches.Surface can further define about 200,000 hole, each holeDiameter is about 50 μm.Alternatively, surface can define about 800,000 micropore, the diameter of each micropore is about 25 μm.Corona can be usedCorona treatment keeps its more hydrophilic to handle surface.
The microorganism (such as bacterium) separated from soil can be diluted to soil extract nutrients, and be applied to chip surfaceWith.It is then possible to chip surface application film.For example, film can be hydrogel layer.But use for example lamination by film it is reversible orIrreversibly it is connected or fixed to chip.Then, second device first can be placed on film and chip surface, by chip surfaceIt is divided into subregion.Hole or micropore in each subregion on surface with some subgroups.
In adaptation on chip, the nutrient medium from environment can be added in each subregion.In incubation period, Ke YijianChip is surveyed to observe bacterial growth, such as detect with the difference in cell growth micropore using optical system.It can be at any timeBetween slowly adjust nutrient medium, until it be can be used for laboratory environment grow bacterium full preparation culture medium.
In adaptation on chip, any hole for wherein observing bacterial growth or micropore can be sampled.It can be by bacteriumSample is transferred to, such as plate, the second chip or conventional tool such as culture dish, to adapt to the culture medium prepared.
Some embodiments can be used for Optimal Medium.For example, the bacterium of single type can be applied to chip surface.SoIt afterwards, can be to chip surface application film.For example, film can be hydrogel layer.But using for example lamination is reversible by film or can notIt is connected or fixed to chip inversely.Then, second device can be placed on film and/or chip surface, by film and/or chipSurface is divided into subregion.Hole or micropore in film and/or each subregion on surface with some subgroups, below film and/or surfaceAbove.Different culture medium (for example, being originated from varying environment) can be added in each subregion.Incubation period can monitor chip to observeGrowth rate in the hole with Different Nutrition preparation or micropore.
Some embodiments can be used for screening the microorganism of dissolving phosphoric acid salt, in particular for agricultural application.Phosphorus is plantMain required macronutrient.Soil phophorus can be managed to optimize crop yield, and to soil application in the form of phosphate fertilizerPhosphorus.However, the soluble inorganic phosphate for being largely applied to soil as chemical fertilizer is fixed rapidly, make plant can not benefitWith.Phosphorus bacteria fertilizer bacterial strain can hydrolyze organic phosphorus and Phos from insoluble compound, and can be used for improving plant growth and productionAmount.Sample including microorganism can be originated from environment, such as (especially effectively hydrolyze from the insoluble compound in environment has soilMachine phosphorus and Phos).According to an embodiment, sample can be loaded to the base of the experimental considerations unit at least one arrayOn the surface at bottom, so that at least some microorganisms occupy multiple experimental considerations units.It can be in multiple realities with various forms of phosphorusIt is incubated for the microorganism occupied in verification certificate member, and compares/screen the ability that they hydrolyze various forms of phosphorus.
Some embodiments can be used for screening the mutant having compared with Seedling height rate.Microorganism (such as bacterium) sample can be withFrom the single laboratory cultures object handled with mutagens.According to an embodiment, sample can be loaded to hasOn the surface of the substrate of the experimental considerations unit of at least one array, so that at least some bacteriums occupy multiple experimental considerations units.It can beIt is incubated for the bacterium occupied in multiple experimental considerations units at least one nutrients, and observes/compares to identify and have compared with Gao ShengThe experimental considerations unit of long rate.
Some embodiments can be used for identifying for example new antibiotic, antifungal agent or antiviral compound.Including micro- lifeThe sample of object (such as bacterium) can be originated from environment.According to an embodiment, sample can be loaded to at least oneOn the surface of the substrate of the experimental considerations unit of array, so that at least some bacteriums occupy multiple experimental considerations units.In multiple experimental considerations unitsMiddle to grow the bacterium occupied for a period of time, then occupying one layer of indicator bacteria of growth on bacterium first, (this may need agar thinLayer).The experimental considerations unit that speculum is generated in indicator bacteria layer shows to generate antibiotic.
Some embodiments can be used for screening specific enzymatic activity.It according to an embodiment, can will include cellSample is loaded on the surface of the substrate of the experimental considerations unit at least one array, so that at least one cell occupies multiple realitiesVerification certificate member.The cell occupied is grown in multiple experimental considerations units for a period of time, is then added and is generated color when being digested and cuttingSubstrate.The experimental considerations unit for generating this color reaction shows required enzymatic activity.
Some embodiments, which can be used for screening, executes biological prosthetic microorganism, in particular for clean environment application.It is dirtyDye and environmental pollution are complicated.For example, the influence that can be discharged into environment of oil spilling or other Naphtha solvents depend on it is many becauseElement.In marine environment, the type of oily type, the temperature of water and coastline influences to clean.Sample including microorganism canTo come from environment, such as the rock of oil-containing is formed.According to an embodiment, sample can be loaded to at least one battle arrayOn the surface of the substrate of the experimental considerations unit of column, so that at least some microorganisms occupy multiple experimental considerations units.There can be differenceIt is incubated for the microorganism occupied in multiple experimental considerations units of type of oil, and compares/screen the ability that they remove or neutralize oil.
Conclusion
While characterized as and illustrate various invention embodiments, those skilled in the art are readily apparent that various other sidesFormula and/or structure execute function as described herein and/or obtain result as described herein and/or one or more advantages, thisEach of kind variant and/or modification are considered in the range of invention embodiment as described herein.More generally, this field skillArt personnel are it will be readily understood that all parameters, size, material and construction as described herein are intended to be exemplary, actual parameter, rulerVery little, material and/or construction will depend on using the specific one or more application of the teachings of the present invention.Those skilled in the art willIt recognizes or is able to use no more than routine experiment and determine many equivalents of specific invention embodiment as described herein.It will thus be appreciated that foregoing embodiments only indicate by way of example, and in the range of the following claims and their equivalentsInterior, invention embodiment can carry out in addition to specifically describing and is claimed.The invention embodiment of the disclosure is claimedEach individually feature, system, object, material, kit and/or method as described herein.In addition, if this feature, beingSystem, product, material, kit and/or method do not have conflicting, two or more this features, system, product, material, examinationAny combination of agent box and/or method includes in the invention scope of the disclosure.
Also, various concept of the invention, which can be embodied in, provides one or more methods of example.One as methodThe movement that part carries out can sort in any suitable manner.Therefore, it can construct and wherein be executed with the sequence for being different from diagramThe embodiment of movement, including some movements are performed simultaneously, although being in an exemplary embodiment sequentially-operating.
The full text of all publications, patent application, patent and other bibliography that are mentioned above is incorporated by reference intoHerein.
Being defined for defining and use herein should be understood as that control dictionary defines, in the document being incorporated by reference intoDefinition and/or the ordinary meaning for defining term.
The indefinite article " one " used in the specification and in the claims unless otherwise clear explanation and "one" should manageSolution is "at least one".
The phrase "and/or" used in the specification and in the claims is construed as referring to element " any one of connectionOr both ", that is, element combines in some cases to be existed, and is respectively present in the case of other.The multiple members enumerated with "and/or"Element should understand in the same manner, that is, in conjunction with " one or more " element.In addition to the element particularly pointed out with "and/or" attributeOutside, other elements are optionally present, no matter it is related or uncorrelated to the element that those are particularly pointed out.Therefore, as non-limitingExample, when with open language for example " comprising " is used together when, " A and/or B " in one embodiment, only refer to A (optionallyInclude the element in addition to B)；In another embodiment, only refer to B (optionally comprising the element in addition to A)；Also at anotherIn embodiment, refer to both A and B (optionally comprising other elements)；Etc..
As used in the specification and in the claims, "or" should be understood as thering is identical meanings with above-mentioned "and/or".ExampleSuch as, when separating the project in list, "or" or "and/or" should be construed as include, that is, include a large amount of or series of elementsAt least one, but also comprise more than one, and other optional unlisted projects.Only when term is explicitly indicated it is opposite when,Such as " only one " or " only one " or " by ... form " refer to (when used in a claim) only comprising a large amount of orOne element of series of elements.In general, be exclusiveness term (such as " any one ", " one of them ", " only one " beforeOr " only one ") when, the term as used herein "or" should be understood merely as indicating exclusiveness selection (that is, " one or anotherBoth it is a, but be not ").When used in a claim, " substantially by ... form " should have it in Patent Law fieldCommon meaning when use.
As used in the specification and in the claims, when referring to the list of one or more elements, phrase " at least oneIt is a " it is understood to refer at least one element selected from any one or more elements of the element list, and it is differentIt surely include each element specifically enumerated in element list, and any combination for the element being not excluded in element list.This is fixedJustice also allows to be optionally present the element in addition to the element particularly pointed out in the element list of phrase "at least one" meaning no matterIts to those of to particularly point out element related or uncorrelated.Therefore, as non-limiting examples, " at least one of A and B "(or equally, " at least one of A or B ", or equally, " at least one of A and/or B ") in one embodiment,Refer at least one (optionally includes more than one) A and (and optional includes element in addition to B) is not present in B；In another realityIt applies in scheme, refers at least one (optionally includes more than one) B and (and optional includes element in addition to A) is not present in A；AlsoIn another embodiment, refer at least one (optionally including more than one) A and at least one (optionally includes more than oneIt is a) B (optionally including other elements)；Etc..
In claim and description above, all transition phrases for example "comprising", " comprising ", " carrying ", " having "," containing ", " being related to ", " receiving ", " by ... constitute ", " defining " etc. be interpreted as it is open, that is, refer to including but not limited to.Only transition phrase " by ... form " and " substantially by ... form " closed or semi-closed transitional should be interpreted as respectivelyPhrase, such as U.S. Patent Office, patent examining procedure handbook, shown in the 2111.03rd trifle.